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THE MYSTERY AND MAGIC OF GLIA

We are interested in the development and function of glial cells in the mammalian central nervous system. To understand the interactions between neurons and glial cells we have developed methods to highly purify and culture retinal ganglion cells (neurons) as well as the glial cell types they interact with, oligodendrocytes and astrocytes, from the rodent optic nerve. We are using a large variety of methods to address these issues including cell purification by immunopanning, tissue culture, patch clamping, immunohistochemistry and molecular biology. Currently, we are focusing on several questions:

(1) What are the cell-cell interactions that control myelination and node of Ranvier formation?

(2) Do glial cells play a role in synapse formation and function?

(3) What are the signals that promote the survival and growth of retinal ganglion cells and can we use this knowledge to promote their survival and regeneration after injury?

(4) How do protoplasmic astrocytes, the main glial cell type in gray matter, develop and what is their function?.

We have found evidence of several novel glial signals that induce the onset of myelination, the clustering of axonal sodium channels, the survival and growth of retinal ganglion cells, and the formation of synapses. We are characterizing these processes and are attempting to identify these glial-derived molecules.

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Novel allele-dependent role for APOE in controlling the rate of synapse pruning by astrocytes.Proceedings of the National Academy of Sciences of the United States of AmericaChung, W., Verghese, P. B., Chakraborty, C., Joung, J., Hyman, B. T., Ulrich, J. D., Holtzman, D. M., Barres, B. A.2016; 113 (36): 10186-10191

Abstract

The strongest genetic risk factor influencing susceptibility to late-onset Alzheimer's disease (AD) is apolipoprotein E (APOE) genotype. APOE has three common isoforms in humans, E2, E3, and E4. The presence of two copies of the E4 allele increases risk by ∼12-fold whereas E2 allele is associated with an ∼twofold decreased risk for AD. These data put APOE central to AD pathophysiology, but it is not yet clear how APOE alleles modify AD risk. Recently we found that astrocytes, a major central nervous system cell type that produces APOE, are highly phagocytic and participate in normal synapse pruning and turnover. Here, we report a novel role for APOE in controlling the phagocytic capacity of astrocytes that is highly dependent on APOE isoform. APOE2 enhances the rate of phagocytosis of synapses by astrocytes, whereas APO4 decreases it. We also found that the amount of C1q protein accumulation in hippocampus, which may represent the accumulation of senescent synapses with enhanced vulnerability to complement-mediated degeneration, is highly dependent on APOE alleles: C1q accumulation was significantly reduced in APOE2 knock-in (KI) animals and was significantly increased in APOE4 KI animals compared with APOE3 KI animals. These studies reveal a novel allele-dependent role for APOE in regulating the rate of synapse pruning by astrocytes. They also suggest the hypothesis that AD susceptibility of APOE4 may originate in part from defective phagocytic capacity of astrocytes which accelerates the rate of accumulation of C1q-coated senescent synapses, enhancing synaptic vulnerability to classical-complement-cascade mediated neurodegeneration.

Abstract

Synapse loss in Alzheimer's disease (AD) correlates with cognitive decline. Involvement of microglia and complement in AD has been attributed to neuroinflammation, prominent late in disease. Here we show in mouse models that complement and microglia mediate synaptic loss early in AD. C1q, the initiating protein of the classical complement cascade, is increased and associated with synapses before overt plaque deposition. Inhibition of C1q, C3, or the microglial complement receptor CR3 reduces the number of phagocytic microglia, as well as the extent of early synapse loss. C1q is necessary for the toxic effects of soluble β-amyloid (Aβ) oligomers on synapses and hippocampal long-term potentiation. Finally, microglia in adult brains engulf synaptic material in a CR3-dependent process when exposed to soluble Aβ oligomers. Together, these findings suggest that the complement-dependent pathway and microglia that prune excess synapses in development are inappropriately activated and mediate synapse loss in AD.

Abstract

The specific function of microglia, the tissue resident macrophages of the brain and spinal cord, has been difficult to ascertain because of a lack of tools to distinguish microglia from other immune cells, thereby limiting specific immunostaining, purification, and manipulation. Because of their unique developmental origins and predicted functions, the distinction of microglia from other myeloid cells is critically important for understanding brain development and disease; better tools would greatly facilitate studies of microglia function in the developing, adult, and injured CNS. Here, we identify transmembrane protein 119 (Tmem119), a cell-surface protein of unknown function, as a highly expressed microglia-specific marker in both mouse and human. We developed monoclonal antibodies to its intracellular and extracellular domains that enable the immunostaining of microglia in histological sections in healthy and diseased brains, as well as isolation of pure nonactivated microglia by FACS. Using our antibodies, we provide, to our knowledge, the first RNAseq profiles of highly pure mouse microglia during development and after an immune challenge. We used these to demonstrate that mouse microglia mature by the second postnatal week and to predict novel microglial functions. Together, we anticipate these resources will be valuable for the future study and understanding of microglia in health and disease.

Abstract

The functional and molecular similarities and distinctions between human and murine astrocytes are poorly understood. Here, we report the development of an immunopanning method to acutely purify astrocytes from fetal, juvenile, and adult human brains and to maintain these cells in serum-free cultures. We found that human astrocytes have abilities similar to those of murine astrocytes in promoting neuronal survival, inducing functional synapse formation, and engulfing synaptosomes. In contrast to existing observations in mice, we found that mature human astrocytes respond robustly to glutamate. Next, we performed RNA sequencing of healthy human astrocytes along with astrocytes from epileptic and tumor foci and compared these to human neurons, oligodendrocytes, microglia, and endothelial cells (available at http://www.brainrnaseq.org). With these profiles, we identified novel human-specific astrocyte genes and discovered a transcriptome-wide transformation between astrocyte precursor cells and mature post-mitotic astrocytes. These data represent some of the first cell-type-specific molecular profiles of the healthy and diseased human brain.

Abstract

Astrocytes are central nervous system (CNS) glial cells with many important functions for normal development and neural functioning. They help control extracellular ion and neurotransmitter concentrations; provide neurotrophic support; are implicated in synapse formation, function, and pruning; and help maintain the blood-brain barrier. Following injury and in disease, they undergo rapid and chronic alterations in function that can either promote or hinder recovery, depending on the disease.

Local axonal protection by WldS as revealed by conditional regulation of protein stability.Proceedings of the National Academy of Sciences of the United States of AmericaWang, J. T., Medress, Z. A., Vargas, M. E., Barres, B. A.2015; 112 (33): 10093-10100

Abstract

The expression of the mutant Wallerian degeneration slow (WldS) protein significantly delays axonal degeneration from various nerve injuries and in multiple species; however, the mechanism for its axonal protective property remains unclear. Although WldS is localized predominantly in the nucleus, it also is present in a smaller axonal pool, leading to conflicting models to account for the WldS fraction necessary for axonal protection. To identify where WldS activity is required to delay axonal degeneration, we adopted a method to alter the temporal expression of WldS protein in neurons by chemically regulating its protein stability. We demonstrate that continuous WldS activity in the axonal compartment is both necessary and sufficient to delay axonal degeneration. Furthermore, by specifically increasing axonal WldS expression postaxotomy, we reveal a critical period of 4-5 h postinjury during which the course of Wallerian axonal degeneration can be halted. Finally, we show that NAD(+), the metabolite of WldS/nicotinamide mononucleotide adenylyltransferase enzymatic activity, is sufficient and specific to confer WldS-like axon protection and is a likely molecular mediator of WldS axon protection. The results delineate a therapeutic window in which the course of Wallerian degeneration can be delayed even after injures have occurred and help narrow the molecular targets of WldS activity to events within the axonal compartment.

Local axonal protection by WldS as revealed by conditional regulation of protein stabilityPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAWang, J. T., Medress, Z. A., Vargas, M. E., Barres, B. A.2015; 112 (33): 10093-10100

Abstract

Myelin is essential in vertebrates for the rapid propagation of action potentials, but the molecular mechanisms driving its formation remain largely unknown. Here we show that the initial stage of process extension and axon ensheathment by oligodendrocytes requires dynamic actin filament assembly by the Arp2/3 complex. Unexpectedly, subsequent myelin wrapping coincides with the upregulation of actin disassembly proteins and rapid disassembly of the oligodendrocyte actin cytoskeleton and does not require Arp2/3. Inducing loss of actin filaments drives oligodendrocyte membrane spreading and myelin wrapping in vivo, and the actin disassembly factor gelsolin is required for normal wrapping. We show that myelin basic protein, a protein essential for CNS myelin wrapping whose role has been unclear, is required for actin disassembly, and its loss phenocopies loss of actin disassembly proteins. Together, these findings provide insight into the molecular mechanism of myelin wrapping and identify it as an actin-independent form of mammalian cell motility.

Abstract

The human cerebral cortex develops through an elaborate succession of cellular events that, when disrupted, can lead to neuropsychiatric disease. The ability to reprogram somatic cells into pluripotent cells that can be differentiated in vitro provides a unique opportunity to study normal and abnormal corticogenesis. Here, we present a simple and reproducible 3D culture approach for generating a laminated cerebral cortex-like structure, named human cortical spheroids (hCSs), from pluripotent stem cells. hCSs contain neurons from both deep and superficial cortical layers and map transcriptionally to in vivo fetal development. These neurons are electrophysiologically mature, display spontaneous activity, are surrounded by nonreactive astrocytes and form functional synapses. Experiments in acute hCS slices demonstrate that cortical neurons participate in network activity and produce complex synaptic events. These 3D cultures should allow a detailed interrogation of human cortical development, function and disease, and may prove a versatile platform for generating other neuronal and glial subtypes in vitro.

Abstract

The human cerebral cortex develops through an elaborate succession of cellular events that, when disrupted, can lead to neuropsychiatric disease. The ability to reprogram somatic cells into pluripotent cells that can be differentiated in vitro provides a unique opportunity to study normal and abnormal corticogenesis. Here, we present a simple and reproducible 3D culture approach for generating a laminated cerebral cortex-like structure, named human cortical spheroids (hCSs), from pluripotent stem cells. hCSs contain neurons from both deep and superficial cortical layers and map transcriptionally to in vivo fetal development. These neurons are electrophysiologically mature, display spontaneous activity, are surrounded by nonreactive astrocytes and form functional synapses. Experiments in acute hCS slices demonstrate that cortical neurons participate in network activity and produce complex synaptic events. These 3D cultures should allow a detailed interrogation of human cortical development, function and disease, and may prove a versatile platform for generating other neuronal and glial subtypes in vitro.

Abstract

The human brain is a tissue of vast complexity in terms of the cell types it comprises. Conventional approaches to classifying cell types in the human brain at single cell resolution have been limited to exploring relatively few markers and therefore have provided a limited molecular characterization of any given cell type. We used single cell RNA sequencing on 466 cells to capture the cellular complexity of the adult and fetal human brain at a whole transcriptome level. Healthy adult temporal lobe tissue was obtained during surgical procedures where otherwise normal tissue was removed to gain access to deeper hippocampal pathology in patients with medical refractory seizures. We were able to classify individual cells into all of the major neuronal, glial, and vascular cell types in the brain. We were able to divide neurons into individual communities and show that these communities preserve the categorization of interneuron subtypes that is typically observed with the use of classic interneuron markers. We then used single cell RNA sequencing on fetal human cortical neurons to identify genes that are differentially expressed between fetal and adult neurons and those genes that display an expression gradient that reflects the transition between replicating and quiescent fetal neuronal populations. Finally, we observed the expression of major histocompatibility complex type I genes in a subset of adult neurons, but not fetal neurons. The work presented here demonstrates the applicability of single cell RNA sequencing on the study of the adult human brain and constitutes a first step toward a comprehensive cellular atlas of the human brain.

Abstract

Alternative splicing (AS) dramatically expands the complexity of the mammalian brain transcriptome, but its atlas remains incomplete. Here we performed deep mRNA sequencing of mouse cortex to discover and characterize alternative exons with potential functional significance. Our analysis expands the list of AS events over 10-fold compared with previous annotations, demonstrating that 72% of multiexon genes express multiple splice variants in this single tissue. To evaluate functionality of the newly discovered AS events, we conducted comprehensive analyses on central nervous system (CNS) cell type-specific splicing, targets of tissue- or cell type-specific RNA binding proteins (RBPs), evolutionary selection pressure, and coupling of AS with nonsense-mediated decay (AS-NMD). We show that newly discovered events account for 23-42% of all cassette exons under tissue- or cell type-specific regulation. Furthermore, over 7,000 cassette exons are under evolutionary selection for regulated AS in mammals, 70% of which are new. Among these are 3,058 highly conserved cassette exons, including 1,014 NMD exons that may function directly to control gene expression levels. These NMD exons are particularly enriched in RBPs including splicing factors and interestingly also regulators for other steps of RNA metabolism. Unexpectedly, a second group of NMD exons reside in genes encoding chromatin regulators. Although the conservation of NMD exons in RBPs frequently extends into lower vertebrates, NMD exons in chromatin regulators are introduced later into the mammalian lineage, implying the emergence of a novel mechanism coupling AS and epigenetics. Our results highlight previously uncharacterized complexity and evolution in the mammalian brain transcriptome.

Abstract

The major cell classes of the brain differ in their developmental processes, metabolism, signaling, and function. To better understand the functions and interactions of the cell types that comprise these classes, we acutely purified representative populations of neurons, astrocytes, oligodendrocyte precursor cells, newly formed oligodendrocytes, myelinating oligodendrocytes, microglia, endothelial cells, and pericytes from mouse cerebral cortex. We generated a transcriptome database for these eight cell types by RNA sequencing and used a sensitive algorithm to detect alternative splicing events in each cell type. Bioinformatic analyses identified thousands of new cell type-enriched genes and splicing isoforms that will provide novel markers for cell identification, tools for genetic manipulation, and insights into the biology of the brain. For example, our data provide clues as to how neurons and astrocytes differ in their ability to dynamically regulate glycolytic flux and lactate generation attributable to unique splicing of PKM2, the gene encoding the glycolytic enzyme pyruvate kinase. This dataset will provide a powerful new resource for understanding the development and function of the brain. To ensure the widespread distribution of these datasets, we have created a user-friendly website (http://web.stanford.edu/group/barres_lab/brain_rnaseq.html) that provides a platform for analyzing and comparing transciption and alternative splicing profiles for various cell classes in the brain.

Abstract

The regenerative capacity of the peripheral nervous system declines with age. Why this occurs, however, is unknown. We demonstrate that 24-month-old mice exhibit an impairment of functional recovery after nerve injury compared to 2-month-old animals. We find no difference in the intrinsic growth capacity between aged and young sensory neurons in vitro or in their ability to activate growth-associated transcriptional programs after injury. Instead, using age-mismatched nerve transplants in vivo, we show that the extent of functional recovery depends on the age of the nerve graft, and not the age of the host. Molecular interrogation of the sciatic nerve reveals that aged Schwann cells (SCs) fail to rapidly activate a transcriptional repair program after injury. Functionally, aged SCs exhibit impaired dedifferentiation, myelin clearance, and macrophage recruitment. These results suggest that the age-associated decline in axonal regeneration results from diminished Schwann cell plasticity, leading to slower myelin clearance.

Abstract

Mammalian central nervous system neurons fail to regenerate after injury or disease, in part due to a progressive loss in intrinsic axon growth ability after birth. Whether lost axon growth ability is due to limited growth resources or to changes in the axonal growth cone is unknown.Static and time-lapse images of purified retinal ganglion cells (RGCs) were analyzed for axon growth rate and growth cone morphology and dynamics without treatment and after manipulating Kruppel-like transcription factor (KLF) expression or applying mechanical tension.Retinal ganglion cells undergo a developmental switch in growth cone dynamics that mirrors the decline in postnatal axon growth rates, with increased filopodial adhesion and decreased lamellar protrusion area in postnatal axonal growth cones. Moreover, expressing growth-suppressive KLF4 or growth-enhancing KLF6 transcription factors elicits similar changes in postnatal growth cones that correlate with axon growth rates. Postnatal RGC axon growth rate is not limited by an inability to achieve axon growth rates similar to embryonic RGCs; indeed, postnatal axons support elongation rates up to 100-fold faster than postnatal axonal growth rates. Rather, the intrinsic capacity for rapid axon growth is due to both growth cone pausing and retraction, as well as to a slightly decreased ability to achieve rapid instantaneous rates of forward progression. Finally, we observed that RGC axon and dendrite growth are regulated independently in vitro.Together, these data support the hypothesis that intrinsic axon growth rate is regulated by an axon-specific growth program that differentially regulates growth cone motility.

Abstract

Brain endothelial cells form a paracellular and transcellular barrier to many blood-borne solutes via tight junctions (TJs) and scarce endocytotic vesicles. The blood-brain barrier (BBB) plays a pivotal role in the healthy and diseased CNS. BBB damage after ischemic stroke contributes to increased mortality, yet the contributions of paracellular and transcellular mechanisms to this process in vivo are unknown. We have created a transgenic mouse strain whose endothelial TJs are labeled with eGFP and have imaged dynamic TJ changes and fluorescent tracer leakage across the BBB in vivo, using two-photon microscopy in the t-MCAO stroke model. Although barrier function is impaired as early as 6 hr after stroke, TJs display profound structural defects only after 2 days. Conversely, the number of endothelial caveolae and transcytosis rate increase as early as 6 hr after stroke. Therefore, stepwise impairment of transcellular followed by paracellular barrier mechanisms accounts for the BBB deficits in stroke.

Abstract

Myelination of the central nervous system requires the generation of functionally mature oligodendrocytes from oligodendrocyte precursor cells (OPC). Electrically active neurons may influence OPC function and selectively instruct myelination of an active neural circuit. Here, we use optogenetic stimulation of premotor cortex in awake, behaving mice to demonstrate that neuronal activity elicits a mitogenic response of neural progenitor cells and OPCs, promotes oligodendrogenesis and increases myelination within the deep layers of the premotor cortex and subcortical white matter. We further show that this neuronal activity-regulated oligodendrogenesis and myelination is associated with improved motor function of the corresponding limb. Oligodendrogenesis and myelination appear necessary for the observed functional improvement, as epigenetic blockade of oligodendrocyte differentiation and myelin changes prevents the activity-regulated behavioral improvement.

Abstract

Enabling axon regeneration after central nervous system (CNS) injury remains a major challenge in neurobiology. One of the major differences between the injured peripheral nervous system (PNS) and CNS is the pro- and antiregenerative responses of their glial cell populations. In addition to intrinsic qualities of the neurons themselves, glial-driven changes to the neural environment have a significant impact on regenerative outcome. This Review presents a comparison of the glial response to injury between the CNS and PNS and highlights features of the PNS glial response that, with continued study, might reveal long-sought-after keys to achieving CNS repair.

Abstract

Astrocytes constitute a major cell population in the brain with a myriad of essential functions, yet we know remarkably little about the signaling pathways and mechanisms that direct astrocyte maturation. To explore the signals regulating astrocyte development, we prospectively purified and cultured immature postnatal rodent astrocytes. We identified fibroblast growth factors (FGFs) and bone morphogenetic proteins (BMPs) as robust trophic factors for immature astrocytes. We showed that astrocytes respond directly to BMPs via phosphorylation of the smad1/5/8 pathway. In vitro, BMP signaling promoted immature astrocytes to adopt multiple characteristics of mature astrocytes, including a more process-bearing morphology, aquaporin-4 (AQP4) and S100β immunoreactivity, limited proliferation, and strong downregulation of epidermal growth factor receptor (EGFR). In vivo, activation of the smad1/5/8 pathway in astrocytes was seen during early postnatal development, but inhibition of astrocyte proliferation was not observed. These insights can aid in the further dissection of the mechanisms and pathways controlling astrocyte biology and development.

Abstract

Oligodendrocytes (OLs) are the myelinating glia of the central nervous system. Myelin is essential for the rapid propagation of action potentials as well as for metabolic support of axons, and its loss in demyelinating diseases like multiple sclerosis has profound pathological consequences. The many steps in the development of OLs - from the specification of oligodendrocyte precursor cells (OPCs) during embryonic development to their differentiation into OLs that myelinate axons - are under tight regulation. Here we discuss recent advances in understanding how these steps of OL development are controlled intrinsically by transcription factors and chromatin remodeling and extrinsically by signaling molecules and neuronal activity. We also discuss how knowledge of these pathways is now allowing us to take steps toward generating patient-specific OPCs for disease modeling and myelin repair.

Abstract

What is the biological basis for human cognition? Our understanding why human brains make us smarter than other animals is still in its infancy. In recent years, astrocytes have been shown to be indispensable for neuronal survival, growth, synapse formation, and synapse function. Now, in a new study from Maiken Nedergaard and Steven Goldman's groups (Han et al., 2013), human glia progenitor cells have been transplanted into mouse forebrains. These progenitors survived, migrated widely, and gave rise to astrocytes that displayed the characteristics of human astrocytes in the rodent host brains. Strikingly, the mice with transplanted human cells displayed improved long term potentiation (LTP) and learning, suggesting the potential importance of human astrocytes in the unique cognitive abilities of human brains. This landmark paper is an important first step toward future investigations of whether and how human astrocytes play a role in distinguishing the cognitive abilities of humans from those of other animals.

Abstract

The myelination of axons is a crucial step during vertebrate central nervous system (CNS) development, allowing for rapid and energy efficient saltatory conduction of nerve impulses. Accordingly, the differentiation of oligodendrocytes, the myelinating cells of the CNS, and their expression of myelin genes are under tight transcriptional control. We previously identified a putative transcription factor, Myelin Regulatory Factor (Myrf), as being vital for CNS myelination. Myrf is required for the generation of CNS myelination during development and also for its maintenance in the adult. It has been controversial, however, whether Myrf directly regulates transcription, with reports of a transmembrane domain and lack of nuclear localization. Here we show that Myrf is a membrane-associated transcription factor that undergoes an activating proteolytic cleavage to separate its transmembrane domain-containing C-terminal region from a nuclear-targeted N-terminal region. Unexpectedly, this cleavage event occurs via a protein domain related to the autoproteolytic intramolecular chaperone domain of the bacteriophage tail spike proteins, the first time this domain has been found to play a role in eukaryotic proteins. Using ChIP-Seq we show that the N-terminal cleavage product directly binds the enhancer regions of oligodendrocyte-specific and myelin genes. This binding occurs via a defined DNA-binding consensus sequence and strongly promotes the expression of target genes. These findings identify Myrf as a novel example of a membrane-associated transcription factor and provide a direct molecular mechanism for its regulation of oligodendrocyte differentiation and CNS myelination.

Abstract

The wiring of the nervous system requires that axons navigate to the correct targets and maintain their correct positions during developmental growth. In this issue, Shao et al. (2013) now reveal a crucial new role for glia in preserving correct synaptic connectivity during developmental growth.

Abstract

Oligodendrocytes-the myelin-forming cells of the central nervous system-can be regenerated during adulthood. In adults, new oligodendrocytes originate from oligodendrocyte progenitor cells (OPCs), but also from neural stem cells (NSCs). Although several factors supporting oligodendrocyte production have been characterized, the mechanisms underlying the generation of adult oligodendrocytes are largely unknown. Here we show that genetic inactivation of SIRT1, a protein deacetylase implicated in energy metabolism, increases the production of new OPCs in the adult mouse brain, in part by acting in NSCs. New OPCs produced following SIRT1 inactivation differentiate normally, generating fully myelinating oligodendrocytes. Remarkably, SIRT1 inactivation ameliorates remyelination and delays paralysis in mouse models of demyelinating injuries. SIRT1 inactivation leads to the upregulation of genes involved in cell metabolism and growth factor signalling, in particular PDGF receptor α (PDGFRα). Oligodendrocyte expansion following SIRT1 inactivation is mediated at least in part by AKT and p38 MAPK-signalling molecules downstream of PDGFRα. The identification of drug-targetable enzymes that regulate oligodendrocyte regeneration in adults could facilitate the development of therapies for demyelinating injuries and diseases, such as multiple sclerosis.

Abstract

Transplantation of oligodendrocyte precursor cells (OPCs) is a promising potential therapeutic strategy for diseases affecting myelin. However, the derivation of engraftable OPCs from human pluripotent stem cells has proven difficult and primary OPCs are not readily available. Here we report the generation of induced OPCs (iOPCs) by direct lineage conversion. Forced expression of the three transcription factors Sox10, Olig2 and Zfp536 was sufficient to reprogram mouse and rat fibroblasts into iOPCs with morphologies and gene expression signatures resembling primary OPCs. More importantly, iOPCs gave rise to mature oligodendrocytes that could ensheath multiple host axons when co-cultured with primary dorsal root ganglion cells and formed myelin after transplantation into shiverer mice. We propose direct lineage reprogramming as a viable alternative approach for the generation of OPCs for use in disease modeling and regenerative medicine.

Abstract

Astrocytes are now emerging as key participants in many aspects of brain development, function and disease. In particular, new evidence shows that astrocytes powerfully control the formation, maturation, function and elimination of synapses through various secreted and contact-mediated signals. Astrocytes are also increasingly being implicated in the pathophysiology of many psychiatric and neurological disorders that result from synaptic defects. A better understanding of how astrocytes regulate neural circuit development and function in the healthy and diseased brain might lead to the development of therapeutic agents to treat these diseases.

Abstract

Microglia are resident immune cells in the brain and spinal cord. These cells provide immune surveillance and are mobilized in response to disparate diseases and injuries. Although microglial activation is often considered neurotoxic, microglia are essential defenders against many neurodegenerative diseases. It also seems increasingly likely that microglial dysfunction can underlie certain neurological diseases without an obvious immune component.

Abstract

Gabapentin (GBP) is an anticonvulsant that acts at the ?2?-1 submit of the L-type calcium channel. It is recently reported that GBP is a potent inhibitor of thrombospondin (TSP)-induced excitatory synapse formation in vitro and in vivo. Here we studied effects of chronic GBP administration on epileptogenesis in the partial cortical isolation ("undercut") model of posttraumatic epilepsy, in which abnormal axonal sprouting and aberrant synaptogenesis contribute to occurrence of epileptiform discharges. Results showed that 1) the incidence of evoked epileptiform discharges in undercut cortical slices studied 1 day or ~2 weeks after the last GBP dose, was significantly reduced by GBP treatments, beginning on the day of injury; 2) the expression of GFAP and TSP1 protein, as well as the number of FJC stained cells was decreased in GBP treated undercut animals; 3) in vivo GBP treatment of rats with undercuts for 3 or 7 days decreased the density of vGlut1-PSD95 close appositions (presumed synapses) in comparison to saline treated controls with similar lesions;4) the electrophysiological data are compatible with the above anatomical changes, showing decreases in mEPSC and sEPSC frequency in the GBP treated animals. These results indicate that chronic administration of GBP after cortical injury is antiepileptogenic in the undercut model of post-traumatic epilepsy, perhaps by both neuroprotective actions and decreases in excitatory synapse formation. The findings may suggest the potential use of GBP as an antiepileptogenic agent following traumatic brain injury.

Abstract

Complement proteins C1q and C3 play a critical role in synaptic elimination during development. Axotomy of spinal motoneurons triggers removal of synaptic terminals from the cell surface of motoneurons by largely unknown mechanisms. We therefore hypothesized that the complement system is involved also in synaptic stripping of injured motoneurons. In the sciatic motor pool of wild type (WT) mice, the immunoreactivity (IR) for both C1q and C3 was increased after sciatic nerve transection (SNT). Mice deficient in C3 (C3(-/-)) showed a reduced loss of synaptic terminals from injured motoneurons at one week after SNT, as assessed by immunoreactivity for synaptic markers and electron microscopy. In particular, the removal of putative inhibitory terminals, immunopositive for vesicular inhibitory amino acid transporter (VIAAT) and ultrastructurally identified as type F synapses, was reduced in C3(-/-) mice. In contrast, lesion-induced removal of nerve terminals in C1q(-/-) mice appeared similar to WT mice. Growth associated protein (GAP)-43 mRNA expression in lesioned motoneurons increased much more in C3(-/-) compared to WT mice after SNT. After sciatic nerve crush (SNC), the C3(-/-) mice showed a faster functional recovery, assessed as grip strength, compared to WT mice. No differences were detected regarding nerve inflammation at the site of injury or pattern of muscle reinnervation. These data indicate that a non-classical pathway of complement activation is involved in axotomy-induced adult synapse removal, and that its inhibition promotes functional recovery.

Abstract

Expansion of astrocyte populations in the central nervous system is characteristic of evolutionarily more complex organisms. However, regulation of mammalian astrocyte precursor proliferation during development remains poorly understood. Here, we used Aldh1L1-GFP to identify two morphologically distinct types of proliferative astrocyte precursors: radial glia (RG) in the ventricular zone and a second cell type we call an 'intermediate astrocyte precursor' (IAP) located in the mantle region of the spinal cord. Astrogenic RG and IAP cells proliferated in a progressive ventral-to-dorsal fashion in a tight window from embryonic day 13.5 until postnatal day 3, which correlated precisely with the pattern of active ERK signalling. Conditional loss of BRAF function using BLBP-cre resulted in a 20% decrease in astrocyte production, whereas expression of activated BRAFV600E resulted in astrocyte hyperproliferation. Interestingly, BRAFV600E mitogenic effects in astrocytes were restricted, in part, by the function of p16INK4A-p19(ARF), which limited the temporal epoch for proliferation. Together, these findings suggest that astrocyte precursor proliferation involves distinct RG and IAP cells; is subjected to temporal and spatial control; and depends in part on BRAF signalling at early stages of mammalian spinal cord development.

Abstract

Neuropathic pain is a common cause of pain after nerve injury, but its molecular basis is poorly understood. In a post-gene chip microarray effort to identify new target genes contributing to neuropathic pain development, we report here the characterization of a novel neuropathic pain contributor, thrombospondin-4 (TSP4), using a neuropathic pain model of spinal nerve ligation injury. TSP4 is mainly expressed in astrocytes and significantly upregulated in the injury side of dorsal spinal cord that correlates with the development of neuropathic pain states. TSP4 blockade by intrathecal antibodies, antisense oligodeoxynucleotides, or inactivation of the TSP4 gene reverses or prevents behavioral hypersensitivities. Intrathecal injection of TSP4 protein into naive rats is sufficient to enhance the frequency of EPSCs in spinal dorsal horn neurons, suggesting an increased excitatory presynaptic input, and to cause similar behavioral hypersensitivities. Together, these findings support that injury-induced spinal TSP4 may contribute to spinal presynaptic hypersensitivity and neuropathic pain states. Development of TSP4 antagonists has the therapeutic potential for target-specific neuropathic pain management.

Abstract

In the developing central nervous system (CNS), the control of synapse number and function is critical to the formation of neural circuits. We previously demonstrated that astrocyte-secreted factors powerfully induce the formation of functional excitatory synapses between CNS neurons. Astrocyte-secreted thrombospondins induce the formation of structural synapses, but these synapses are postsynaptically silent. Here we use biochemical fractionation of astrocyte-conditioned medium to identify glypican 4 (Gpc4) and glypican 6 (Gpc6) as astrocyte-secreted signals sufficient to induce functional synapses between purified retinal ganglion cell neurons, and show that depletion of these molecules from astrocyte-conditioned medium significantly reduces its ability to induce postsynaptic activity. Application of Gpc4 to purified neurons is sufficient to increase the frequency and amplitude of glutamatergic synaptic events. This is achieved by increasing the surface level and clustering, but not overall cellular protein level, of the GluA1 subunit of the AMPA (?-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) glutamate receptor (AMPAR). Gpc4 and Gpc6 are expressed by astrocytes in vivo in the developing CNS, with Gpc4 expression enriched in the hippocampus and Gpc6 enriched in the cerebellum. Finally, we demonstrate that Gpc4-deficient mice have defective synapse formation, with decreased amplitude of excitatory synaptic currents in the developing hippocampus and reduced recruitment of AMPARs to synapses. These data identify glypicans as a family of novel astrocyte-derived molecules that are necessary and sufficient to promote glutamate receptor clustering and receptivity and to induce the formation of postsynaptically functioning CNS synapses.

Abstract

Excessive synapses generated during early development are eliminated extensively to form functionally mature neural circuits. Synapses in juvenile and mature brains are highly dynamic, and undergo remodeling processes through constant formation and elimination of dendritic spines. Although neural activity has been implicated in initiating the synapse elimination process cell-autonomously, the cellular and molecular mechanisms that transduce changes in correlated neural activity into structural changes in synapses are largely unknown. Recently, however, new findings provide evidence that in different species, glial cells, non-neuronal cell types in the nervous system are crucial in eliminating neural debris and unwanted synapses through phagocytosis. Glial cells not only clear fragmented axons and synaptic debris produced during synapse elimination, but also engulf unwanted synapses thereby actively promoting synapse elimination non-cell autonomously. These new findings support the important role of glial cells in the formation and maintenance of functional neural circuits in development as well as in adult stages and neurodegenerative diseases.

Abstract

Microglia are the resident CNS immune cells and active surveyors of the extracellular environment. While past work has focused on the role of these cells during disease, recent imaging studies reveal dynamic interactions between microglia and synaptic elements in the healthy brain. Despite these intriguing observations, the precise function of microglia at remodeling synapses and the mechanisms that underlie microglia-synapse interactions remain elusive. In the current study, we demonstrate a role for microglia in activity-dependent synaptic pruning in the postnatal retinogeniculate system. We show that microglia engulf presynaptic inputs during peak retinogeniculate pruning and that engulfment is dependent upon neural activity and the microglia-specific phagocytic signaling pathway, complement receptor 3(CR3)/C3. Furthermore, disrupting microglia-specific CR3/C3 signaling resulted in sustained deficits in synaptic connectivity. These results define a role for microglia during postnatal development and identify underlying mechanisms by which microglia engulf and remodel developing synapses.

Abstract

Reactive astrogliosis is characterized by a profound change in astrocyte phenotype in response to all CNS injuries and diseases. To better understand the reactive astrocyte state, we used Affymetrix GeneChip arrays to profile gene expression in populations of reactive astrocytes isolated at various time points after induction using two mouse injury models, ischemic stroke and neuroinflammation. We find reactive gliosis consists of a rapid, but quickly attenuated, induction of gene expression after insult and identify induced Lcn2 and Serpina3n as strong markers of reactive astrocytes. Strikingly, reactive astrocyte phenotype strongly depended on the type of inducing injury. Although there is a core set of genes that is upregulated in reactive astrocytes from both injury models, at least 50% of the altered gene expression is specific to a given injury type. Reactive astrocytes in ischemia exhibited a molecular phenotype that suggests that they may be beneficial or protective, whereas reactive astrocytes induced by LPS exhibited a phenotype that suggests that they may be detrimental. These findings demonstrate that, despite well established commonalities, astrocyte reactive gliosis is a highly heterogeneous state in which astrocyte activities are altered to respond to the specific injury. This raises the question of how many subtypes of reactive astrocytes exist. Our findings provide transcriptome databases for two subtypes of reactive astrocytes that will be highly useful in generating new and testable hypotheses of their function, as well as for providing new markers to detect different types of reactive astrocytes in human neurological diseases.

Abstract

Astrocytes are no longer seen as a homogenous population of cells. In fact, recent studies indicate that astrocytes are morphologically and functionally diverse and play critical roles in neurodevelopmental diseases such as Rett syndrome and fragile X mental retardation. This review summarizes recent advances in astrocyte development, including the role of neural tube patterning in specification and developmental functions of astrocytes during synaptogenesis. We propose here that a precise understanding of astrocyte development is critical to defining heterogeneity and could lead advances in understanding and treating a variety of neuropsychiatric diseases.

Abstract

Hypothyroidism is a well-described cause of hypomyelination. In addition, thyroid hormone (T3) has recently been shown to enhance remyelination in various animal models of CNS demyelination. What are the ways in which T3 promotes the development and regeneration of healthy myelin? To begin to understand the mechanisms by which T3 drives myelination, we have identified genes regulated specifically by T3 in purified oligodendrocyte precursor cells (OPCs). Among the genes identified by genomic expression analyses were four transcription factors, Kruppel-like factor 9 (KLF9), basic helix-loop-helix family member e22 (BHLHe22), Hairless (Hr), and Albumin D box-binding protein (DBP), all of which were induced in OPCs by both brief and long term exposure to T3. To begin to investigate the role of these genes in myelination, we focused on the most rapidly and robustly induced of these, KLF9, and found it is both necessary and sufficient to promote oligodendrocyte differentiation in vitro. Surprisingly, we found that loss of KLF9 in vivo negligibly affects the formation of CNS myelin during development, but does significantly delay remyelination in cuprizone-induced demyelinated lesions. These experiments indicate that KLF9 is likely a novel integral component of the T3-driven signaling cascade that promotes the regeneration of lost myelin. Future analyses of the roles of KLF9 and other identified T3-induced genes in myelination may lead to novel insights into how to enhance the regeneration of myelin in demyelinating diseases such as multiple sclerosis.

Abstract

Expression of the Wld(s) protein significantly delays axon degeneration in injuries and diseases, but the mechanism for this protection is unknown. Two recent reports present evidence that axonal mitochondria are required for Wld(S)-mediated axon protection.

Abstract

The SCN8A gene encodes the voltage-gated sodium channel Na(v)1.6, a major channel in neurons of the CNS and PNS. SCN8A contains two alternative exons,18N and 18A, that exhibit tissue specific splicing. In brain, the major SCN8A transcript contains exon 18A and encodes the full-length sodium channel. In other tissues, the major transcript contains exon 18N and encodes a truncated protein, due to the presence of an in-frame stop codon. Selection of exon 18A is therefore essential for generation of a functional channel protein, but the proteins involved in this selection have not been identified. Using a 2.6 kb Scn8a minigene containing exons 18N and 18A, we demonstrate that co-transfection with Fox-1 or Fox-2 initiates inclusion of exon 18A. This effect is dependent on the consensus Fox binding site located 28 bp downstream of exon 18A. We examined the alternative splicing of human SCN8A and found that the postnatal switch to exon 18A is completed later than 10 months of age. In purified cell populations, transcripts containing exon 18A predominate in neurons but are not present in oligodendrocytes or astrocytes. Transcripts containing exon 18N appear to be degraded by nonsense-mediated decay in HEK cells. Our data indicate that RBFOX proteins contribute to the cell-specific expression of Na(v)1.6 channels in mature neurons.

Abstract

Microglia are the abundant, resident myeloid cells of the central nervous system (CNS) that become rapidly activated in response to injury or inflammation. While most studies of microglia focus on this phenomenon, little is known about the function of 'resting' microglia, which possess fine, branching cellular processes. Biber and colleagues, in a recent paper in Journal of Neuroinflammation, report that ramified microglia can limit excitotoxicity, an important insight for understanding mechanisms that limit neuron death in CNS disease.

Abstract

Axon degeneration is a characteristic event in many neurodegenerative conditions including stroke, glaucoma, and motor neuropathies. However, the molecular pathways that regulate this process remain unclear. Axon loss in chronic neurodegenerative diseases share many morphological features with those in acute injuries, and expression of the Wallerian degeneration slow (WldS) transgene delays nerve degeneration in both events, indicating a common mechanism of axonal self-destruction in traumatic injuries and degenerative diseases. A proposed model of axon degeneration is that nerve insults lead to impaired delivery or expression of a local axonal survival factor, which results in increased intra-axonal calcium levels and calcium-dependent cytoskeletal breakdown.

Abstract

An unexpected role for the classical complement cascade in the elimination of central nervous system (CNS) synapses has recently been discovered. Complement proteins are localized to developing CNS synapses during periods of active synapse elimination and are required for normal brain wiring. The function of complement proteins in the brain appears analogous to their function in the immune system: clearance of cellular material that has been tagged for elimination. Similarly, synapses tagged with complement proteins may be eliminated by microglial cells expressing complement receptors. In addition, developing astrocytes release signals that induce the expression of complement components in the CNS. In the mature brain, early synapse loss is a hallmark of several neurodegenerative diseases. Complement proteins are profoundly upregulated in many CNS diseases prior to signs of neuron loss, suggesting a reactivation of similar developmental mechanisms of complement-mediated synapse elimination potentially driving disease progression.

Abstract

Myelin is a lipid-rich, spiraled membrane structure that allows for rapid propagation of action potentials through axons. In this issue, Aggarwal et al. (2011) present evidence that myelin basic protein, essential for myelination by oligodendrocytes, regulates the biosynthesis of myelin membranes by restricting diffusion of membrane-bound proteins into compact myelin.

Abstract

The inability to purify and culture astrocytes has long hindered studies of their function. Whereas astrocyte progenitor cells can be cultured from neonatal brain, culture of mature astrocytes from postnatal brain has not been possible. Here, we report a new method to prospectively purify astrocytes by immunopanning. These astrocytes undergo apoptosis in culture, but vascular cells and HBEGF promote their survival in serum-free culture. We found that some developing astrocytes normally undergo apoptosis in vivo and that the vast majority of astrocytes contact blood vessels, suggesting that astrocytes are matched to blood vessels by competing for vascular-derived trophic factors such as HBEGF. Compared to traditional astrocyte cultures, the gene profiles of the cultured purified postnatal astrocytes much more closely resemble those of in vivo astrocytes. Although these astrocytes strongly promote synapse formation and function, they do not secrete glutamate in response to stimulation.

Abstract

Neural circuits consist of highly precise connections among specific types of neurons that serve a common functional goal. How neurons distinguish among different synaptic targets to form functionally precise circuits remains largely unknown. Here, we show that during development, the adhesion molecule cadherin-6 (Cdh6) is expressed by a subset of retinal ganglion cells (RGCs) and also by their targets in the brain. All of the Cdh6-expressing retinorecipient nuclei mediate non-image-forming visual functions. A screen of mice expressing GFP in specific subsets of RGCs revealed that Cdh3-RGCs which also express Cdh6 selectively innervate Cdh6-expressing retinorecipient targets. Moreover, in Cdh6-deficient mice, the axons of Cdh3-RGCs fail to properly innervate their targets and instead project to other visual nuclei. These findings provide functional evidence that classical cadherins promote mammalian CNS circuit development by ensuring that axons of specific cell types connect to their appropriate synaptic targets.

Abstract

Astrocytes regulate synaptic connectivity in the CNS through secreted signals. Here we identified two astrocyte-secreted proteins, hevin and SPARC, as regulators of excitatory synaptogenesis in vitro and in vivo. Hevin induces the formation of synapses between cultured rat retinal ganglion cells. SPARC is not synaptogenic, but specifically antagonizes synaptogenic function of hevin. Hevin and SPARC are expressed by astrocytes in the superior colliculus, the synaptic target of retinal ganglion cells, concurrent with the excitatory synaptogenesis. Hevin-null mice had fewer excitatory synapses; conversely, SPARC-null mice had increased synaptic connections in the superior colliculus. Furthermore, we found that hevin is required for the structural maturation of the retinocollicular synapses. These results identify hevin as a positive and SPARC as a negative regulator of synapse formation and signify that, through regulation of relative levels of hevin and SPARC, astrocytes might control the formation, maturation, and plasticity of synapses in vivo.

Abstract

On-Off direction-selective retinal ganglion cells (DSGCs) encode the axis of visual motion. They respond strongly to an object moving in a preferred direction and weakly to an object moving in the opposite, "null," direction. Historically, On-Off DSGCs were classified into four subtypes according to their directional preference (anterior, posterior, superior, or inferior). Here, we compare two genetically identified populations of On-Off DSGCs: dopamine receptor 4 (DRD4)-DSGCs and thyrotropin-releasing hormone receptor (TRHR)-DSGCs. We find that although both populations are tuned for posterior motion, they can be distinguished by a variety of physiological and anatomical criteria. First, the directional tuning of TRHR-DSGCs is broader than that of DRD4-DSGCs. Second, whereas both populations project similarly to the dorsal lateral geniculate nucleus, they project differently to the ventral lateral geniculate nucleus and the superior colliculus. Moreover, TRHR-DSGCs, but not DRD4-DSGCs, also project to the zona incerta, a thalamic area not previously known to receive direction-tuned visual information. Our findings reveal unexpected diversity among mouse On-Off DSGC subtypes that uniquely process and convey image motion to the brain.

Abstract

CNS myelin is strongly inhibitory to growing axons and is thought to be a major contributor to CNS axon regenerative failure. Although a number of proteins present in myelin, including Nogo, MAG, and oligodendrocyte-myelin glycoprotein (OMgp), have been identified as myelin-associated inhibitors, studies of mice lacking these genes suggest that additional inhibitors present in CNS myelin remain to be identified. Here we have investigated the hypothesis that myelin lipids contribute to CNS regenerative failure. We identified sulfatide, a major constituent of CNS myelin, as a novel myelin-associated inhibitor of neurite outgrowth. Sulfatide, but not galactocerebroside or ceramide, strongly inhibited the neurite outgrowth of retinal ganglion cells (RGCs) when used as a purified lipid substrate. The mechanism involved in sulfatide-mediated inhibition may share features with other known inhibitors, because the Rho inhibitor C3 transferase lessened these effects. Myelin in which sulfatide was lacking or blocked using specific antibodies was significantly less inhibitory to RGC neurite outgrowth in vitro than was wild-type myelin, indicating that sulfatide is a major component of the inhibitory activity of CNS myelin. Mice unable to make sulfatide did not regenerate RGC axons more robustly after optic nerve crush than wild-type littermates under normal conditions but did exhibit a small but significant enhancement in the extent of zymosan-induced regeneration. These results demonstrate that specific lipids can powerfully inhibit axon growth, identify sulfatide as a novel myelin-associated axon growth inhibitor, and provide evidence that sulfatide inhibition contributes to axon regenerative failure in vivo.

Abstract

Down syndrome (DS) is a developmental disorder caused by a third chromosome 21 in humans (Trisomy 21), leading to neurological deficits and cognitive impairment. Studies in mouse models of DS suggest that cognitive deficits in the adult are associated with deficits in synaptic learning and memory mechanisms, but it is unclear whether alterations in the early wiring and refinement of neuronal circuits contribute to these deficits. Here, we show that early developmental refinement of visual circuits is perturbed in mouse models of Down syndrome. Specifically, we find excessive eye-specific segregation of retinal axons in the dorsal lateral geniculate nucleus. Indeed, the degree of refinement scales with defects in the "Down syndrome critical region" (DSCR) in a dose-dependent manner. We further identify Dscam (Down syndrome cell adhesion molecule), a gene within the DSCR, as a regulator of eye-specific segregation of retinogeniculate projections. Although Dscam is not the sole gene in the DSCR contributing to enhanced refinement in trisomy, Dscam dosage clearly regulates cell spacing and dendritic fasciculation in a specific class of retinal ganglion cells. Thus, altered developmental refinement of visual circuits that occurs before sensory experience is likely to contribute to visual impairment in individuals with Down syndrome.

Abstract

Differentiation of oligodendrocyte progenitor cells (OPCs) into mature oligodendrocytes requires extensive changes in gene expression, which are partly mediated by post-translational modifications of nucleosomal histones. An essential modification for oligodendrocyte differentiation is the removal of acetyl groups from lysine residues which is catalyzed by histone deacetylases (HDACs). The transcriptional targets of HDAC activity within OPCs however, have remained elusive and have been identified in this study by interrogating the oligodendrocyte transcriptome. Using a novel algorithm that allows clustering of gene transcripts according to expression kinetics and expression levels, we defined major waves of co-regulated genes. The initial overall decrease in gene expression was followed by the up-regulation of genes involved in lipid metabolism and myelination. Functional annotation of the down-regulated gene clusters identified transcripts involved in cell cycle regulation, transcription, and RNA processing. To define whether these genes were the targets of HDAC activity, we cultured rat OPCs in the presence of trichostatin A (TSA), an HDAC inhibitor previously shown to inhibit oligodendrocyte differentiation. By overlaying the defined oligodendrocyte transcriptome with the list of 'TSA sensitive' genes, we determined that a high percentage of 'TSA sensitive' genes are part of a normal program of oligodendrocyte differentiation. TSA treatment increased the expression of genes whose down-regulation occurs very early after induction of OPC differentiation, but did not affect the expression of genes with a slower kinetic. Among the increased 'TSA sensitive' genes we detected several transcription factors including Id2, Egr1, and Sox11, whose down-regulation is critical for OPC differentiation. Thus, HDAC target genes include clusters of co-regulated genes involved in transcriptional repression. These results support a de-repression model of oligodendrocyte lineage progression that relies on the concurrent down-regulation of several inhibitors of differentiation.

Abstract

Glaucoma is one of the most common neurodegenerative diseases. Despite this, the earliest stages of this complex disease are still unclear. This study was specifically designed to identify early stages of glaucoma in DBA/2J mice. To do this, we used genome-wide expression profiling of optic nerve head and retina and a series of computational methods. Eyes with no detectable glaucoma by conventional assays were grouped into molecularly defined stages of disease using unbiased hierarchical clustering. These stages represent a temporally ordered sequence of glaucoma states. We then determined networks and biological processes that were altered at these early stages. Early-stage expression changes included upregulation of both the complement cascade and the endothelin system, and so we tested the therapeutic value of separately inhibiting them. Mice with a mutation in complement component 1a (C1qa) were protected from glaucoma. Similarly, inhibition of the endothelin system with bosentan, an endothelin receptor antagonist, was strongly protective against glaucomatous damage. Since endothelin 2 is potently vasoconstrictive and was produced by microglia/macrophages, our data provide what we believe to be a novel link between these cell types and vascular dysfunction in glaucoma. Targeting early molecular events, such as complement and endothelin induction, may provide effective new treatments for human glaucoma.

Abstract

Throughout the nervous system, neurons restrict their connections to specific depths or "layers" of their targets to constrain the type and number of synapses they make. Despite the importance of lamina-specific synaptic connectivity, the mechanisms that give rise to this feature in mammals remain poorly understood. Here we examined the cellular events underlying the formation of lamina-specific retinal ganglion cell (RGC) axonal projections to the superior colliculus (SC) of the mouse. By combining a genetically encoded marker of a defined RGC subtype (OFF-?RGCs) with serial immunoelectron microscopy, we resolved the ultrastructure of axon terminals fated for laminar stabilization versus those fated for removal. We found that OFF-?RGCs form synapses across the full depth of the retinorecipient SC before undergoing lamina-specific arbor retraction and synapse elimination to arrive at their mature, restricted pattern of connectivity. Interestingly, we did not observe evidence of axon degeneration or glia-induced synapse engulfment during this process. These findings indicate that lamina-specific visual connections are generated through the selective stabilization of correctly targeted axon arbors and suggest that the decision to maintain or eliminate an axonal projection reflects the molecular compatibility of presynaptic and postsynaptic neurons at a given laminar depth.

Abstract

Vascular endothelial cells in the central nervous system (CNS) form a barrier that restricts the movement of molecules and ions between the blood and the brain. This blood-brain barrier (BBB) is crucial to ensure proper neuronal function and protect the CNS from injury and disease. Transplantation studies have demonstrated that the BBB is not intrinsic to the endothelial cells, but is induced by interactions with the neural cells. Owing to the close spatial relationship between astrocytes and endothelial cells, it has been hypothesized that astrocytes induce this critical barrier postnatally, but the timing of BBB formation has been controversial. Here we demonstrate that the barrier is formed during embryogenesis as endothelial cells invade the CNS and pericytes are recruited to the nascent vessels, over a week before astrocyte generation. Analysing mice with null and hypomorphic alleles of Pdgfrb, which have defects in pericyte generation, we demonstrate that pericytes are necessary for the formation of the BBB, and that absolute pericyte coverage determines relative vascular permeability. We demonstrate that pericytes regulate functional aspects of the BBB, including the formation of tight junctions and vesicle trafficking in CNS endothelial cells. Pericytes do not induce BBB-specific gene expression in CNS endothelial cells, but inhibit the expression of molecules that increase vascular permeability and CNS immune cell infiltration. These data indicate that pericyte-endothelial cell interactions are critical to regulate the BBB during development, and disruption of these interactions may lead to BBB dysfunction and neuroinflammation during CNS injury and disease.

Abstract

The human brain contains more than 100 trillion (10(14)) synaptic connections, which form all of its neural circuits. Neuroscientists have long been interested in how this complex synaptic web is weaved during development and remodelled during learning and disease. Recent studies have uncovered that glial cells are important regulators of synaptic connectivity. These cells are far more active than was previously thought and are powerful controllers of synapse formation, function, plasticity and elimination, both in health and disease. Understanding how signalling between glia and neurons regulates synaptic development will offer new insight into how the nervous system works and provide new targets for the treatment of neurological diseases.

Abstract

The blood-brain barrier (BBB) maintains brain homeostasis and limits the entry of toxins and pathogens into the brain. Despite its importance, little is known about the molecular mechanisms regulating the development and function of this crucial barrier. In this study we have developed methods to highly purify and gene profile endothelial cells from different tissues, and by comparing the transcriptional profile of brain endothelial cells with those purified from the liver and lung, we have generated a comprehensive resource of transcripts that are enriched in the BBB forming endothelial cells of the brain. Through this comparison we have identified novel tight junction proteins, transporters, metabolic enzymes, signaling components, and unknown transcripts whose expression is enriched in central nervous system (CNS) endothelial cells. This analysis has identified that RXRalpha signaling cascade is specifically enriched at the BBB, implicating this pathway in regulating this vital barrier. This dataset provides a resource for understanding CNS endothelial cells and their interaction with neural and hematogenous cells.

Abstract

Astrocytes, one of the most numerous types of cells in the central nervous system, are crucial for potassium homeostasis, neurotransmitter uptake, synapse formation, regulation of blood-brain-barrier, and the development of the nervous system. Historically, astrocytes have been studied as a homogeneous group of cells. However, evidence has accumulated that suggests heterogeneity of astrocytes across brain regions as well as within the same brain regions. Astrocytes differ in their morphology, developmental origin, gene expression profile, physiological properties, function, and response to injury and disease. A better understanding of the heterogeneity of astrocytes will greatly aid investigation of the function of astrocytes in normal brain as well as the roles of astrocytes in neurological disorders.

Abstract

Degenerating myelin inhibits axon regeneration and is rapidly cleared after peripheral (PNS) but not central nervous system (CNS) injury. To better understand mechanisms underlying rapid PNS myelin clearance, we tested the potential role of the humoral immune system. Here, we show that endogenous antibodies are required for rapid and robust PNS myelin clearance and axon regeneration. B-cell knockout JHD mice display a significant delay in macrophage influx, myelin clearance, and axon regeneration. Rapid clearance of myelin debris is restored in mutant JHD mice by passive transfer of antibodies from naïve WT mice or by an anti-PNS myelin antibody, but not by delivery of nonneural antibodies. We demonstrate that degenerating nerve tissue is targeted by preexisting endogenous antibodies that control myelin clearance by promoting macrophage entrance and phagocytic activity. These results demonstrate a role for immunoglobulin (Ig) in clearing damaged self during healing and suggest that the immune-privileged status of the CNS may contribute to failure of CNS myelin clearance and axon regeneration after injury.

Abstract

Hereditary hypotrichosis simplex is a rare autosomal dominant form of hair loss characterized by hair follicle miniaturization. Using genetic linkage analysis, we mapped a new locus for the disease to chromosome 18p11.22, and identified a mutation (Leu9Arg) in the adenomatosis polyposis down-regulated 1 (APCDD1) gene in three families. We show that APCDD1 is a membrane-bound glycoprotein that is abundantly expressed in human hair follicles, and can interact in vitro with WNT3A and LRP5-two essential components of Wnt signalling. Functional studies show that APCDD1 inhibits Wnt signalling in a cell-autonomous manner and functions upstream of beta-catenin. Moreover, APCDD1 represses activation of Wnt reporters and target genes, and inhibits the biological effects of Wnt signalling during both the generation of neurons from progenitors in the developing chick nervous system, and axis specification in Xenopus laevis embryos. The mutation Leu9Arg is located in the signal peptide of APCDD1, and perturbs its translational processing from the endoplasmic reticulum to the plasma membrane. APCDD1(L9R) probably functions in a dominant-negative manner to inhibit the stability and membrane localization of the wild-type protein. These findings describe a novel inhibitor of the Wnt signalling pathway with an essential role in human hair growth. As APCDD1 is expressed in a broad repertoire of cell types, our findings indicate that APCDD1 may regulate a diversity of biological processes controlled by Wnt signalling.

Abstract

To investigate the role of microRNAs in regulating oligodendrocyte (OL) differentiation and myelination, we utilized transgenic mice in which microRNA processing was disrupted in OL precursor cells (OPCs) and OLs by targeted deletion of Dicer1. We found that inhibition of OPC-OL miRNA processing disrupts normal CNS myelination and that OPCs lacking mature miRNAs fail to differentiate normally in vitro. We identified three miRNAs (miR-219, miR-138, and miR-338) that are induced 10-100x during OL differentiation; the most strongly induced of these, miR-219, is necessary and sufficient to promote OL differentiation, and partially rescues OL differentiation defects caused by total miRNA loss. miR-219 directly represses the expression of PDGFRalpha, Sox6, FoxJ3, and ZFP238 proteins, all of which normally help to promote OPC proliferation. Together, these findings show that miR-219 plays a critical role in coupling differentiation to proliferation arrest in the OL lineage, enabling the rapid transition from proliferating OPCs to myelinating OLs.

Abstract

The controlling factors that prompt mature oligodendrocytes to myelinate axons are largely undetermined. In this study, we used a forward genetics approach to identify a mutant mouse strain characterized by the absence of CNS myelin despite the presence of abundant numbers of late-stage, process-extending oligodendrocytes. Through linkage mapping and complementation testing, we identified the mutation as a single nucleotide insertion in the gene encoding zinc finger protein 191 (Zfp191), which is a widely expressed, nuclear-localized protein that belongs to a family whose members contain both DNA-binding zinc finger domains and protein-protein-interacting SCAN domains. Zfp191 mutants express an array of myelin-related genes at significantly reduced levels, and our in vitro and in vivo data indicate that mutant ZFP191 acts in a cell-autonomous fashion to disrupt oligodendrocyte function. Therefore, this study demonstrates that ZFP191 is required for the myelinating function of differentiated oligodendrocytes.

Abstract

Synapses are asymmetric cellular adhesions that are critical for nervous system development and function, but the mechanisms that induce their formation are not well understood. We have previously identified thrombospondin as an astrocyte-secreted protein that promotes central nervous system (CNS) synaptogenesis. Here, we identify the neuronal thrombospondin receptor involved in CNS synapse formation as alpha2delta-1, the receptor for the anti-epileptic and analgesic drug gabapentin. We show that the VWF-A domain of alpha2delta-1 interacts with the epidermal growth factor-like repeats common to all thrombospondins. alpha2delta-1 overexpression increases synaptogenesis in vitro and in vivo and is required postsynaptically for thrombospondin- and astrocyte-induced synapse formation in vitro. Gabapentin antagonizes thrombospondin binding to alpha2delta-1 and powerfully inhibits excitatory synapse formation in vitro and in vivo. These findings identify alpha2delta-1 as a receptor involved in excitatory synapse formation and suggest that gabapentin may function therapeutically by blocking new synapse formation.

Abstract

A primer on new research by Fuentes-Medel and colleagues explains the important role of non-neural cells in clearing neural debris, which is continuously produced during the normal remodeling processes that establish and maintain neural connectivity.

Abstract

The transcriptional control of CNS myelin gene expression is poorly understood. Here we identify gene model 98, which we have named myelin gene regulatory factor (MRF), as a transcriptional regulator required for CNS myelination. Within the CNS, MRF is specifically expressed by postmitotic oligodendrocytes. MRF is a nuclear protein containing an evolutionarily conserved DNA binding domain homologous to a yeast transcription factor. Knockdown of MRF in oligodendrocytes by RNA interference prevents expression of most CNS myelin genes; conversely, overexpression of MRF within cultured oligodendrocyte progenitors or the chick spinal cord promotes expression of myelin genes. In mice lacking MRF within the oligodendrocyte lineage, premyelinating oligodendrocytes are generated but display severe deficits in myelin gene expression and fail to myelinate. These mice display severe neurological abnormalities and die because of seizures during the third postnatal week. These findings establish MRF as a critical transcriptional regulator essential for oligodendrocyte maturation and CNS myelination.

Abstract

Motion detection is an essential component of visual processing. On-Off direction-selective retinal ganglion cells (On-Off DSGCs) detect objects moving along specific axes of the visual field due to their precise retinal circuitry. The brain circuitry of On-Off DSGCs, however, is largely unknown. We report a mouse with GFP expressed selectively by the On-Off DSGCs that detect posterior motion (On-Off pDSGCs), allowing two-photon targeted recordings of their light responses and delineation of their complete map of central connections. On-Off pDSGCs project exclusively to the dorsal lateral geniculate nucleus and superior colliculus and in both targets form synaptic lamina that are separate from a lamina corresponding to non-DSGCs. Thus, individual On-Off DSGC subtypes are molecularly distinct and establish circuits that map specific qualities of directional motion to dedicated subcortical areas. This suggests that each RGC subtype represents a unique parallel pathway whose synaptic specificity in the retina is recapitulated in central targets.

Wnt/beta-catenin signaling is required for CNS, but not non-CNS, angiogenesisPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICADaneman, R., Agalliu, D., Zhou, L., Kuhnert, F., Kuo, C. J., Barres, B. A.2009; 106 (2): 641-646

Abstract

Despite the importance of CNS blood vessels, the molecular mechanisms that regulate CNS angiogenesis and blood-brain barrier (BBB) formation are largely unknown. Here we analyze the role of Wnt/beta-catenin signaling in regulating the formation of CNS blood vessels. First, through the analysis of TOP-Gal Wnt reporter mice, we identify that canonical Wnt/beta-catenin signaling is specifically activated in CNS, but not non-CNS, blood vessels during development. This activation correlates with the expression of different Wnt ligands by neural progenitor cells in distinct locations throughout the CNS, including Wnt7a and Wnt7b in ventral regions and Wnt1, Wnt3, Wnt3a, and Wnt4 in dorsal regions. Blockade of Wnt/beta-catenin signaling in vivo specifically disrupts CNS, but not non-CNS, angiogenesis. These defects include reduction in vessel number, loss of capillary beds, and the formation of hemorrhagic vascular malformations that remain adherent to the meninges. Furthermore, we demonstrate that Wnt/beta-catenin signaling regulates the expression of the BBB-specific glucose transporter glut-1. Taken together these experiments reveal an essential role for Wnt/beta-catenin signaling in driving CNS-specific angiogenesis and provide molecular evidence that angiogenesis and BBB formation are in part linked.

Abstract

Mechanistic studies of CNS myelination have been hindered by the lack of a rapidly myelinating culture system. Here, we describe a versatile CNS coculture method that allows time-lapse microscopy and molecular analysis of distinct stages of myelination. Employing a culture architecture of reaggregated neurons fosters extension of dense beds of axons from purified retinal ganglion cells. Seeding of oligodendrocyte precursor cells on these axons results in differentiation and ensheathment in as few as 3 days, with generation of compact myelin within 6 days. This technique enabled (1) the demonstration that oligodendrocytes initiate new myelin segments only during a brief window early in their differentiation, (2) identification of a contribution of astrocytes to the rate of myelin wrapping, and (3) molecular dissection of the role of oligodendrocyte gamma-secretase activity in controlling the ensheathment of axons. These insights illustrate the value of this defined system for investigating multiple aspects of CNS myelination.

Abstract

A major challenge to understanding how cells work together in the central nervous system (CNS) is the heterogeneous cellular composition of the brain. In this issue, Heiman et al. (2008) and Doyle et al. (2008) introduce a new strategy (TRAP) that enables the profiling of translated mRNAs in specific CNS cell populations without the need for purifying cells to homogeneity.

The Mystery and Magic of Glia: A Perspective on Their Roles in Health and DiseaseNEURONBarres, B. A.2008; 60 (3): 430-440

Abstract

In this perspective, I review recent evidence that glial cells are critical participants in every major aspect of brain development, function, and disease. Far more active than once thought, glial cells powerfully control synapse formation, function, and blood flow. They secrete many substances whose roles are not understood, and they are central players in CNS injury and disease. I argue that until the roles of nonneuronal cells are more fully understood and considered, neurobiology as a whole will progress only slowly.

Abstract

Aspartoacylase (ASPA) is an oligodendrocyte-restricted enzyme that catalyzes the hydrolysis of neuronally derived N-acetylaspartate (NAA) to acetate and aspartic acid. ASPA deficiency leads to the fatal childhood autosomal recessive leukodystrophy Canavan disease (CD). Here we demonstrate that the previously described ENU-induced nur7 mouse mutant is caused by a nonsense mutation, Q193X, in the Aspa gene (Aspa(nur7)). Homozygous Aspa(nur7nur7) mice do not express detectable Aspa protein and display an early-onset spongy degeneration of CNS myelin with increased NAA levels similar to that observed in CD patients. In addition, CNS regions rich in neuronal cell bodies also display vacuolization. Interestingly, distinct myelin rich areas, such as the corpus callosum, optic nerve, and spinal cord white matter appear normal in Aspa(nur7/nur7) mice. Reduced cerebroside synthesis has been demonstrated in CD patients and animal models. To determine the potential relevance of this observation in disease pathogenesis, we generated Aspa(nur7/nur7) mice that were heterozygous for a null allele of the gene that encodes the enzyme UDP-galactose:ceramide galactosyltransferase (Cgt), which is responsible for catalyzing the synthesis of the abundant myelin galactolipids. Despite reduced amounts of cerebrosides, the Aspa(nur7/nur7);Cgt(+/-) mice were not more severely affected than the Aspa(nur7) mutants, suggesting that diminished cerebroside synthesis is not a major contributing factor in disease pathogenesis. Furthermore, we found that myelin degeneration leads to significant axonal loss in the cerebellum of older Aspa(nur7) mutants. This finding suggests that axonal pathology caused by CNS myelin defects may underlie the neurological disabilities that CD patients develop at late stages of the disease.

Abstract

Our understanding of how mammalian sensory circuits are organized and develop has long been hindered by the lack of genetic markers of neurons with discrete functions. Here, we report a transgenic mouse selectively expressing GFP in a complete mosaic of transient OFF-alpha retinal ganglion cells (tOFF-alphaRGCs). This enabled us to relate the mosaic spacing, dendritic anatomy, and electrophysiology of these RGCs to their complete map of projections in the brain. We find that tOFF-alphaRGCs project exclusively to the superior colliculus (SC) and dorsal lateral geniculate nucleus and are restricted to a specific laminar depth within each of these targets. The axons of tOFF-alphaRGC are also organized into columns in the SC. Both laminar and columnar specificity develop through axon refinement. Disruption of cholinergic retinal waves prevents the emergence of columnar- but not laminar-specific tOFF-alphaRGC connections. Our findings reveal that in a genetically identified sensory map, spontaneous activity promotes synaptic specificity by segregating axons arising from RGCs of the same subtype.

Abstract

One of the difficulties in studying cellular interactions in the CNS is the lack of effective methods to purify specific neuronal populations of interest. We report the development of a novel purification scheme, cholera toxin beta (CTB) immunopanning, in which a particular CNS neuron population is selectively labeled via retrograde axonal transport of the cell-surface epitope CTB, and then purified via immobilization with anti-CTB antibody. We have demonstrated the usefulness and versatility of this method by purifying both retinal ganglion cells and corticospinal motor neurons (CSMNs). Genomic expression analyses of purified CSMNs revealed that they express significant levels of many receptors for growth factors produced by brain endothelial cells; three of these factors, CXCL12, pleiotrophin, and IGF2 significantly enhanced purified CSMN survival, similar to previously characterized CSMN trophic factors BDNF and IGF1. In addition, endothelial cell conditioned medium significantly promoted CSMN neurite outgrowth. These findings demonstrate a useful method for the purification of several different types of CNS projection neurons, which in principle should work in many mammalian species, and provide evidence that endothelial-derived factors may represent an overlooked source of trophic support for neurons in the brain.

Abstract

Are neurons born with the ability to form and receive synapses or do they acquire these abilities during development? We have previously found that purified postnatal retinal ganglion cells (RGCs) require soluble astrocyte-derived signals to form synapses in vitro and in vivo. Here we show that newly generated embryonic day 17 (E17) RGCs are able to form but not receive synapses under these conditions. Dendrite growth is not sufficient to trigger receptivity; rather, the ability of newly generated RGCs to receive synapses is acquired at E19 in response to direct contact by neighboring cell types. Direct contact with astrocytes, which are not present at E17 but are normally generated by E19, is sufficient to induce synaptic receptivity in E17 RGCs. In contrast, amacrine contact does not induce synaptic receptivity. Interestingly, astrocyte contact alters the localization of the synaptic adhesion molecule neurexin away from dendrites. In addition, dendritic expression of neurexin is sufficient to prevent astrocyte contact-mediated increases in synapse number, suggesting a molecular mechanism by which astrocyte contact regulates neuronal synaptic receptivity. Thus, synaptic receptivity is not induced simply by dendritic elaboration but must be signaled by both contact-mediated signaling from astrocytes and a shift in the dendritic localization of neurexin.

Abstract

In vitro culturing of primary neurons is a mainstay of neurobiological research. Many of these culture paradigms have taken advantage of defined culture media rather than serum additives that contain undefined survival factors to facilitate experimental manipulations and interpretation of the results. To culture neurons in the absence of serum, defined supplements such as B27 are now widely used. However, commercially available supplements exhibit large variability in their capabilities to support neurons in culture. We re-optimized and modified earlier published formulations of B27 using 21 different ingredients (NS21). NS21 supports neuronal cultures of high quality as manifested by their morphological characteristics, formation of synapses, and postsynaptic responses. Much of the variability in the quality of B27/NS21 was due to variability in the quality of different sources of bovine serum albumin. Furthermore, we found that holo-transferrin used in NS21 is preferable over apo-transferrin used in B27 for the quality of neuronal cultures.

Abstract

Understanding the cell-cell interactions that control CNS development and function has long been limited by the lack of methods to cleanly separate neural cell types. Here we describe methods for the prospective isolation and purification of astrocytes, neurons, and oligodendrocytes from developing and mature mouse forebrain. We used FACS (fluorescent-activated cell sorting) to isolate astrocytes from transgenic mice that express enhanced green fluorescent protein (EGFP) under the control of an S100beta promoter. Using Affymetrix GeneChip Arrays, we then created a transcriptome database of the expression levels of >20,000 genes by gene profiling these three main CNS neural cell types at various postnatal ages between postnatal day 1 (P1) and P30. This database provides a detailed global characterization and comparison of the genes expressed by acutely isolated astrocytes, neurons, and oligodendrocytes. We found that Aldh1L1 is a highly specific antigenic marker for astrocytes with a substantially broader pattern of astrocyte expression than the traditional astrocyte marker GFAP. Astrocytes were enriched in specific metabolic and lipid synthetic pathways, as well as the draper/Megf10 and Mertk/integrin alpha(v)beta5 phagocytic pathways suggesting that astrocytes are professional phagocytes. Our findings call into question the concept of a "glial" cell class as the gene profiles of astrocytes and oligodendrocytes are as dissimilar to each other as they are to neurons. This transcriptome database of acutely isolated purified astrocytes, neurons, and oligodendrocytes provides a resource to the neuroscience community by providing improved cell-type-specific markers and for better understanding of neural development, function, and disease.

Abstract

During development, the formation of mature neural circuits requires the selective elimination of inappropriate synaptic connections. Here we show that C1q, the initiating protein in the classical complement cascade, is expressed by postnatal neurons in response to immature astrocytes and is localized to synapses throughout the postnatal CNS and retina. Mice deficient in complement protein C1q or the downstream complement protein C3 exhibit large sustained defects in CNS synapse elimination, as shown by the failure of anatomical refinement of retinogeniculate connections and the retention of excess retinal innervation by lateral geniculate neurons. Neuronal C1q is normally downregulated in the adult CNS; however, in a mouse model of glaucoma, C1q becomes upregulated and synaptically relocalized in the adult retina early in the disease. These findings support a model in which unwanted synapses are tagged by complement for elimination and suggest that complement-mediated synapse elimination may become aberrantly reactivated in neurodegenerative disease.

Abstract

To what extent do postmitotic neurons regulate gene expression during development or after injury? We took advantage of our ability to highly purify retinal ganglion cells (RGCs) to profile their pattern of gene expression at 13 ages from embryonic day 17 through postnatal day 21. We found that a large proportion of RGC genes are regulated dramatically throughout their postmitotic development, although the genes regulated through development in vivo generally are not regulated similarly by RGCs allowed to age in vitro. Interestingly, we found that genes regulated by developing RGCs are not generally correlated with genes regulated in RGCs stimulated to regenerate their axons. We unexpectedly found three genes associated with glaucoma, optineurin, cochlin, and CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), previously thought to be primarily expressed in the trabecular meshwork, which are highly expressed by RGCs and regulated through their development. We also identified several other RGC genes that are encoded by loci linked to glaucoma. The expression of glaucoma-linked genes by RGCs suggests that, at least in some cases, RGCs may be directly involved in glaucoma pathogenesis rather than indirectly involved in response to increased intraocular pressure. Consistent with this hypothesis, we found that CYP1B1 overexpression potentiates RGC survival.

Abstract

The intracellular molecular mechanism that controls the timing of oligodendrocyte differentiation remains unknown. Temple and Raff (1986) previously showed that an oligodendrocyte precursor cell (OPC) can divide a maximum of approximately eight times before its daughter cells simultaneously cease proliferating and differentiate into oligodendrocytes. They postulated that over time the level of an intracellular molecule might synchronously change in each daughter cell, ultimately reaching a level that prohibited additional proliferation. Here, we report the discovery of such a molecule, the cyclin-dependent kinase inhibitor p57(Kip2) (Cdkn1c). We show in vitro that all daughters of a clone of OPCs express similar levels of p57(Kip2), that p57(Kip2) levels increase over time in proliferating OPCs, and that p57(Kip2) levels regulate how many times an OPC can divide before differentiating. These findings reveal a novel part of the mechanism by which OPCs measure time and are likely to extend to similar timers in many other precursor cell types.

Abstract

Wallerian degeneration (WD) is the set of molecular and cellular events by which degenerating axons and myelin are cleared after injury. Why WD is rapid and robust in the PNS but slow and incomplete in the CNS is a longstanding mystery. Here we review current work on the mechanisms of WD with an emphasis on deciphering this mystery and on understanding whether slow WD in the CNS could account for the failure of CNS axons to regenerate.

Abstract

To better understand the molecular mechanisms governing oligodendrocyte (OL) differentiation, we have used gene profiling to quantitatively analyze gene expression in synchronously differentiating OLs generated from pure oligodendrocyte precursor cells in vitro. By comparing gene expression in these OLs to OLs generated in vivo, we discovered that the program of OL differentiation can progress normally in the absence of heterologous cell-cell interactions. In addition, we found that OL differentiation was unexpectedly prolonged and occurred in at least two sequential stages, each characterized by changes in distinct complements of transcription factors and myelin proteins. By disrupting the normal dynamic expression patterns of transcription factors regulated during OL differentiation, we demonstrated that these sequential stages of gene expression can be independently controlled. We also uncovered several genes previously uncharacterized in OLs that encode transmembrane, secreted, and cytoskeletal proteins that are as highly upregulated as myelin genes during OL differentiation. Last, by comparing genomic loci associated with inherited increased risk of multiple sclerosis (MS) to genes regulated during OL differentiation, we identified several new positional candidate genes that may contribute to MS susceptibility. These findings reveal a previously unexpected complexity to OL differentiation and suggest that an intrinsic program governs successive phases of OL differentiation as these cells extend and align their processes, ensheathe, and ultimately myelinate axons.

Abstract

Neuronal pentraxins (NPs) define a family of proteins that are homologous to C-reactive and acute-phase proteins in the immune system and have been hypothesized to be involved in activity-dependent synaptic plasticity. To investigate the role of NPs in vivo, we generated mice that lack one, two, or all three NPs. NP1/2 knock-out mice exhibited defects in the segregation of eye-specific retinal ganglion cell (RGC) projections to the dorsal lateral geniculate nucleus, a process that involves activity-dependent synapse formation and elimination. Retinas from mice lacking NP1 and NP2 had cholinergically driven waves of activity that occurred at a frequency similar to that of wild-type mice, but several other parameters of retinal activity were altered. RGCs cultured from these mice exhibited a significant delay in functional maturation of glutamatergic synapses. Other developmental processes, such as pathfinding of RGCs at the optic chiasm and hippocampal long-term potentiation and long-term depression, appeared normal in NP-deficient mice. These data indicate that NPs are necessary for early synaptic refinements in the mammalian retina and dorsal lateral geniculate nucleus. We speculate that NPs exert their effects through mechanisms that parallel the known role of short pentraxins outside the CNS.

Abstract

During mammalian neurogenesis, progenitor cells can divide with the mitotic spindle oriented parallel or perpendicular to the surface of the neuroepithelium. Perpendicular divisions are more likely to be asymmetric and generate one progenitor and one neuronal precursor. Whether the orientation of the mitotic spindle actually determines their asymmetric outcome is unclear. Here, we characterize a mammalian homolog of Inscuteable (mInsc), a key regulator of spindle orientation in Drosophila. mInsc is expressed temporally and spatially in a manner that suggests a role in orienting the mitotic spindle in the developing nervous system. Using retroviral RNAi in rat retinal explants, we show that downregulation of mInsc inhibits vertical divisions. This results in enhanced proliferation, consistent with a higher frequency of symmetric divisions generating two proliferating cells. Our results suggest that the orientation of neural progenitor divisions is important for cell fate specification in the retina and determines their symmetric or asymmetric outcome.

Abstract

Despite the importance of the blood-brain barrier (BBB), little is known about the molecular mechanisms that control its integrity. The identification of moody, a gene required for the formation and maintenance of the Drosophila BBB, provides new insight into how paracellular junctions are formed at the barrier. Meanwhile, moody also has been identified in a screen for fly mutants with altered sensitivity to cocaine, remarkably implicating the BBB in the physiological response to narcotics.

Abstract

Glial cells are now emerging from the shadows cast by their more excitable CNS counterparts. Within the developing nervous system, astrocytes and Schwann cells actively help to promote synapse formation and function, and have even been implicated in synapse elimination. In the adult brain, astrocytes respond to synaptic activity by releasing transmitters that modulate synaptic activity. Thus, glia are active participants in brain function. Many questions remain about the identity of glial-neuronal signals and their significance.

Abstract

A novel neuronal receptor complex that mediates myelin's inhibitory action on nerve fiber regeneration has at last been identified. This discovery could be an important step towards promoting nerve regeneration after stroke or spinal cord injury.

Abstract

The establishment of neural circuitry requires vast numbers of synapses to be generated during a specific window of brain development, but it is not known why the developing mammalian brain has a much greater capacity to generate new synapses than the adult brain. Here we report that immature but not mature astrocytes express thrombospondins (TSPs)-1 and -2 and that these TSPs promote CNS synaptogenesis in vitro and in vivo. TSPs induce ultrastructurally normal synapses that are presynaptically active but postsynaptically silent and work in concert with other, as yet unidentified, astrocyte-derived signals to produce functional synapses. These studies identify TSPs as CNS synaptogenic proteins, provide evidence that astrocytes are important contributors to synaptogenesis within the developing CNS, and suggest that TSP-1 and -2 act as a permissive switch that times CNS synaptogenesis by enabling neuronal molecules to assemble into synapses within a specific window of CNS development.

Abstract

Nearly one-half of the cells in a human brain are astrocytes, but the function of these little cells remains a great mystery. Astrocytes form an intimate association with synapses throughout the adult CNS, where they help regulate ion and neurotransmitter concentrations. Recent in vitro studies, however, have found that astrocytes also exert powerful control over the number of CNS synapses that form, are essential for postsynaptic function, and are required for synaptic stability and maintenance. Moreover, recent studies increasingly implicate astrocytes in vivo as participants in activity-dependent structural and functional synaptic changes throughout the nervous system. Taken together, these data force us to rethink the role of glia. We propose that astrocytes should not be viewed primarily as support cells, but rather as cells that actively control the structural and functional plasticity of synapses in developing and adult organisms.

Abstract

NMDA excitotoxicity has been proposed to mediate the death of retinal ganglion cells (RGCs) in glaucoma and ischemia. Here, we reexamine the effects of glutamate and NMDA on rat RGCs in vitro and in situ. We show that highly purified RGCs express NR1 and NR2 receptor subunits by Western blotting and immunostaining, and functional NMDA receptor channels by whole-cell patch-clamp recording. Nevertheless, high concentrations of glutamate or NMDA failed to induce the death of purified RGCs, even after prolonged exposure for 24 h. RGCs co-cultured together with ephrins, astrocytes, or mixed retinal cells were similarly invulnerable to glutamate and NMDA, though their NMDA currents were 4-fold larger. In contrast, even a short exposure to glutamate or NMDA induced the rapid and profound excitotoxic death of most hippocampal neurons in culture. To determine whether RGCs in an intact retina are vulnerable to excitotoxicity, we retrogradely labeled RGCs in vivo using fluorogold and exposed acutely isolated intact retinas to high concentrations of glutamate or NMDA. This produced a substantial and rapid loss of amacrine cells; however, RGCs were not affected. Nonetheless, RGCs expressed NMDA currents in situ that were larger than those reported for amacrine cells. Interestingly, the NMDA receptors expressed by RGCs were extrasynaptically localized both in vitro and in situ. These results indicate that RGCs in vitro and in situ are relatively invulnerable to glutamate and NMDA excitotoxicity compared to amacrine cells, and indicate that important, as yet unidentified, determinants downstream of NMDA receptors control vulnerability to excitotoxicity.

Abstract

Axons dictate whether or not they will become myelinated in both the central and peripheral nervous systems by providing signals that direct the development of myelinating glia. Here we identify the neurotrophin nerve growth factor (NGF) as a potent regulator of the axonal signals that control myelination of TrkA-expressing dorsal root ganglion neurons (DRGs). Unexpectedly, these NGF-regulated axonal signals have opposite effects on peripheral and central myelination, promoting myelination by Schwann cells but reducing myelination by oligodendrocytes. These findings indicate a novel role for growth factors in regulating the receptivity of axons to myelination and reveal that different axonal signals control central and peripheral myelination.

Abstract

Inhibitors associated with CNS myelin are thought to be important in the failure of axons to regenerate after spinal cord injury and in other neurodegenerative disorders. Here we show that targeting the CNS-specific inhibitor of neurite outgrowth Nogo A by active immunization blunts clinical signs, demyelination and axonal damage associated with experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). Mice vaccinated against Nogo A produce Nogo-specific antibodies that block the neurite outgrowth inhibitory activity associated with CNS myelin in vitro. Passive immunization with anti-Nogo IgGs also suppresses EAE. Our results identify Nogo A as an important determinant of the development of EAE and suggest that its blockade may help to maintain and/or to restore the neuronal integrity of the CNS after autoimmune insult in diseases such as MS. Our finding that Nogo A is involved in CNS autoimmune demyelination indicates that this molecule may have a far more complex role than has been previously anticipated.

Abstract

In the mammalian CNS, glial cells repel axons during development and inhibit axon regeneration after injury. It is unknown whether the same repulsive axon guidance molecules expressed by glia and their precursors during development also play a role in inhibiting regeneration in the injured CNS. Here we investigate whether optic nerve glial cells express semaphorin family members and, if so, whether these semaphorins inhibit axon growth by retinal ganglion cells (RGCs). We show that each optic nerve glial cell type, astrocytes, oligodendrocytes, and their precursor cells, expressed a distinct complement of semaphorins. One of these, sema5A, was expressed only by purified oligodendrocytes and their precursors, but not by astrocytes, and was present in both normal and axotomized optic nerve but not in peripheral nerves. Sema5A induced collapse of RGC growth cones and inhibited RGC axon growth when presented as a substrate in vitro. To determine whether sema5A might contribute to inhibition of axon growth after injury, we studied the ability of RGCs to extend axons when cultured on postnatal day (P) 4, P8, and adult optic nerve explants and found that axon growth was strongly inhibited. Blocking sema5A using a neutralizing antibody significantly increased RGC axon growth on these optic nerve explants. These data support the hypothesis that sema5A expression by oligodendrocyte lineage cells contributes to the glial cues that inhibit CNS regeneration.

Abstract

Glia constitute 90% of cells in the human nervous system, but relatively little is known about their functions. We have been focusing on the potential synaptic roles of glia in the CNS. We recently found that astrocytes increase the number of mature, functional synapses on retinal ganglion cells (RGCs) by sevenfold and are required for synaptic maintenance in vitro. These observations raised the question of whether glia similarly enhance synapse formation by other neuron types. Here we have investigated whether highly purified motor neurons isolated from developing rat spinal cords are able to form synapses in the absence of glia or whether glia similarly enhance synapse number. We show that spinal motor neurons (SMNs) form few synapses unless Schwann cells or astrocytes are present. Schwann cells increase the number of functional synapses by ninefold as measured by immunostaining, and increase spontaneous synaptic activity by several hundredfold. Surprisingly, the synapses formed between spinal motor neurons were primarily glutamatergic, as they could be blocked by CNQX. This synapse-promoting activity is not mediated by direct glial-neuronal cell contact but rather is mediated by secreted molecule(s) from the Schwann cells, as we previously found for astrocytes. Interestingly, the synapse-promoting activity from astrocytes and Schwann cells was functionally similar: Schwann cells also promoted synapse formation between retinal ganglion cells, and astrocytes promoted synapse formation between spinal motor neurons. These studies show that both astrocytes and Schwann cells strongly promote synapse formation between spinal motor neurons and demonstrate that glial regulation of synaptogenesis extends to other neuron types.

Abstract

Cell diversification in the developing nervous system is thought to involve both cell-intrinsic mechanisms and extracellular signals, but their relative importance in particular cell fate decisions remains uncertain. In the mammalian retina, different cell types develop on a predictable schedule from multipotent retinal neuroepithelial cells (RNECs). A current view is that RNECs pass through a series of competence states, progressively changing their responsiveness to instructive extracellular cues, which also change over time. We show here, however, that embryonic day 16-17 (E16-17) rat RNECs develop similarly in serum-free clonal-density cultures and in serum-containing retinal explants--in the number of times they divide, the cell types they generate, and the order in which they generate these cell types. These surprising results suggest that extracellular signals may be less important than currently believed in determining when RNECs stop dividing and what cell types they generate when they withdraw from the cell cycle, at least from E16-17 onward.

Abstract

Three different myelin proteins, Nogo, MAG, and OMgp, inhibit regenerating axons after CNS injury. New work reveals that they all share a common receptor and that blockade of this receptor promotes CNS repair and functional recovery.

Abstract

The central nervous system (CNS) loses the ability to regenerate early during development, but it is not known why. The retina has long served as a simple model system for study of CNS regeneration. Here we show that amacrine cells signal neonatal rat retinal ganglion cells (RGCs) to undergo a profound and apparently irreversible loss of intrinsic axon growth ability. Concurrently, retinal maturation triggers RGCs to greatly increase their dendritic growth ability. These results suggest that adult CNS neurons fail to regenerate not only because of CNS glial inhibition but also because of a loss of intrinsic axon growth ability.

Abstract

We investigate the signaling mechanisms that induce retinal ganglion cell (RGC) axon elongation by asking whether surviving neurons extend axons by default. We show that bcl-2 overexpression is sufficient to keep purified RGCs alive in the absence of any glial or trophic support. The bcl-2-expressing RGCs do not extend axons or dendrites unless signaled to do so by single peptide trophic factors. Axon growth stimulated by peptide trophic factors is remarkably slow but is profoundly potentiated by physiological levels of electrical activity spontaneously generated within embryonic explants or mimicked on a multielectrode silicon chip. These findings demonstrate that these surviving neurons do not constitutively extend axons and provide insight into the signals that may be necessary to promote CNS regeneration.

Abstract

Na(v)1.6 is the main sodium channel isoform at adult nodes of Ranvier. Here, we show that Na(v)1.2 and its beta2 subunit, but not Na(v)1.6 or beta1, are clustered in developing central nervous system nodes and that clustering of Na(v)1.2 and Na(v)1.6 is differentially controlled. Oligodendrocyte-conditioned medium is sufficient to induce clustering of Na(v)1.2 alpha and beta2 subunits along central nervous system axons in vitro. This clustering is regulated by electrical activity and requires an intact actin cytoskeleton and synthesis of a non-sodium channel protein. Neither soluble- or contact-mediated glial signals induce clustering of Na(v)1.6 or beta1 in a nonmyelinating culture system. These data reveal that the sequential clustering of Na(v)1.2 and Na(v)1.6 channels is differentially controlled and suggest that myelination induces Na(v)1.6 clustering.

Abstract

Compared to neurons, the intracellular mechanisms that control glial differentiation are still poorly understood. We show here that oligodendrocyte lineage cells express the helix-loop-helix proteins Mash1 and Id2. Although Mash1 has been found to regulate neuronal development, we found that in the absence of Mash1 oligodendrocyte differentiation occurs normally. In contrast, we found that overexpression of Id2 powerfully inhibits oligodendrocyte differentiation, that Id2 normally translocates out of the nucleus at the onset of differentiation, and that absence of Id2 induces premature oligodendrocyte differentiation in vitro. These findings demonstrate that Id2 is a component of the intracellular mechanism that times oligodendrocyte differentiation and point to the existence of an as yet unidentified MyoD-like bHLH protein necessary for oligodendrocyte differentiation.

Abstract

Here we have investigated the mechanisms that control astrocyte differentiation within the developing rat optic nerve. Astrocytes are normally generated by astrocyte precursor cells within the embryonic optic nerve. We show that there is a close temporal and spatial correlation between endothelial and astrocyte differentiation. We tested the potential role of endothelial cells in inducing astrocyte differentiation by developing an immunopanning method to highly purify endothelial cells from developing optic nerves. We show that the purified endothelial cells, but not other embryonic optic nerve cell types, strongly induce the differentiation of purified astrocyte precursor cells into astrocytes in vitro. Leukemia inhibitory factor (LIF) and LIF receptors have been implicated previously in astrocyte differentiation in vivo. We show that purified endothelial cells express LIF mRNA and that their ability to induce astrocyte differentiation is prevented by a neutralizing anti-LIF, but not anti-ciliary neurotrophic factor, antiserum. These findings demonstrate a role for endothelial cells in inducing astrocyte differentiation. The induction of astrocyte differentiation by endothelial cells makes sense phylogenetically, anatomically, and functionally, because astrocytes evolved concurrently with brain vasculature and ensheathe capillaries throughout the brain. The ability to purify and culture astrocytes and endothelial cells should provide an excellent model system for future studies of blood-brain barrier development.

Abstract

Although astrocytes constitute nearly half of the cells in our brain, their function is a long-standing neurobiological mystery. Here we show by quantal analyses, FM1-43 imaging, immunostaining, and electron microscopy that few synapses form in the absence of glial cells and that the few synapses that do form are functionally immature. Astrocytes increase the number of mature, functional synapses on central nervous system (CNS) neurons by sevenfold and are required for synaptic maintenance in vitro. We also show that most synapses are generated concurrently with the development of glia in vivo. These data demonstrate a previously unknown function for glia in inducing and stabilizing CNS synapses, show that CNS synapse number can be profoundly regulated by nonneuronal signals, and raise the possibility that glia may actively participate in synaptic plasticity.

Abstract

Here, we review progress in our understanding of neuronal and glial cell biology during the past ten years, with an emphasis on glial cell fate specification, apoptosis, the cytoskeleton, neuronal polarity, synaptic vesicle recycling and targeting, regulation of the cytoskeleton by extracellular signals, and neuron-glia interactions.

Abstract

The ability of peripheral nervous system (PNS) but not central nervous system (CNS) neurons to regenerate their axons is a striking peculiarity of higher vertebrates. Much research has focused on the inhibitory signals produced by CNS glia that thwart regenerating axons. Less attention has been paid to the injury-induced loss of trophic stimuli needed to promote the survival and regeneration of axotomized neurons. Could differences in the mechanisms that control CNS and PNS neuronal survival and growth also contribute to the disparity in regenerative capacity? Here we review recent studies concerning the nature of the signals necessary to promote neuronal survival and growth, with an emphasis on their significance to regeneration after CNS injury.

Abstract

In order to study the signals that control the onset of myelination, we cocultured highly purified postnatal retinal ganglion cells and optic nerve oligodendrocytes under serum-free conditions that promote their survival for at least a month and found that no myelination occurred. Although the addition of optic nerve astrocytes induced the oligodendrocyte processes to align with, and adhere to, axons, myelination still did not occur. The effect of astrocytes was mimicked by removal of polysialic acid from both cell types using neuroaminidase. These findings provide evidence for a novel role for astrocytes in controlling the onset of myelination by promoting adhesion of oligodendrocyte processes to axons. They also suggest that other, as yet unidentified, cell-cell interactions are necessary to induce the myelination process itself.

Abstract

The signaling interactions that control oligodendrocyte generation from their precursor cells have been studied intensively. Much less is known about how astrocyte generation is normally controlled. Here we report the purification and characterization of astrocyte precursor cells (APCs) from the developing rat optic nerve. APCs are antigenically distinct from astrocytes. Both cell types are Pax2(+) and vimentin+, whereas astrocytes are GFAP+ and S100beta+, and the precursor cells are A2B5(+). In contrast to purified astrocytes, purified APCs rapidly die in serum-free culture but can be saved by basic fibroblast growth factor (bFGF) and glial growth factor 2 (GGF2). Unlike oligodendrocyte precursor cells, APCs do not differentiate by default; their differentiation into GFAP+ cells is induced by ciliary neurotrophic factor (CNTF) or by leukemia inhibitory factor (LIF). Finally, the survival, proliferation, and differentiation of APCs were promoted by coculture with other embryonic optic nerve cell types but not with purified embryonic retinal ganglion cells, indicating that interactions with non-neuronal cells are likely to play an important role in controlling astrocyte generation in the developing optic nerve.

Abstract

The short-term survival of highly purified embryonic spinal motor neurons (SMNs) in culture can be promoted by many peptide trophic factors, including brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), fibroblast growth factor (FGF), glial-derived neurotrophic factor (GDNF), and hepatocyte growth factor (HGF). We have asked whether these peptides are sufficient to promote the long-term survival of purified E15 SMNs. Contrary to previous reports, we find that when SMNs are cultured in serum-free medium containing a single peptide trophic factor only approximately one-third of the cells survive for 3 d in culture. When multiple factors are combined, additive effects on survival are observed transiently, but by 7 d of culture the majority of SMNs has died. Surprisingly, when cAMP levels are elevated, the majority of SMNs extend processes and survive for 1 week in culture in the absence of peptide trophic factors, even in low-density cultures. A combination of five peptide trophic factors, together with cAMP elevation, promotes the long-term survival of most of the SMNs in serum-free culture for 3 weeks. These findings provide useful culture conditions for studying the properties of SMNs and have implications for the treatment of motor neuron diseases.

Abstract

Oligodendrocyte precursor cells (OPCs) persist in substantial numbers in the adult brain in a quiescent state suggesting that they may provide a source of new oligodendrocytes after injury. To determine whether adult OPCs have the capacity to divide rapidly, we have developed a method to highly purify OPCs from adult optic nerve and have directly compared their properties with their perinatal counterparts. When cultured in platelet-derived growth factor (PDGF), an astrocyte-derived mitogen, perinatal OPCs divided approximately once per day, whereas adult OPCs divided only once every 3 or 4 d. The proliferation rate of adult OPCs was not increased by addition of fibroblast growth factor (FGF) or of the neuregulin glial growth factor 2 (GGF2), two mitogens that are normally produced by retinal ganglion cells. cAMP elevation has been shown previously to be essential for Schwann cells to survive and divide in response to GGF2 and other mitogens. Similarly we found that when cAMP levels were elevated, GGF2 alone was sufficient to induce perinatal OPCs to divide slowly, approximately once every 4 d, but adult OPCs still did not divide. When PDGF was combined with GGF2 and cAMP elevation, however, the adult OPCs began to divide rapidly. These findings indicate that adult OPCs are intrinsically different than perinatal OPCs. They are not senescent cells, however, because they retain the capacity to divide rapidly. Thus, after demyelinating injuries, enhanced axonal release of GGF2 or a related neuregulin might collaborate with astrocyte-derived PDGF to induce rapid division of adult OPCs.

Abstract

Many studies have shown that myelin in the central nervous system strongly inhibits the regeneration of axons, so it comes as a surprise to discover that adult neurons transplanted into the brain rapidly extend their axons through myelinated pathways.

Abstract

Recent studies suggest that interactions between neurons, glial cells and endothelial cells are critical in determining the structure of the retina and the optic nerve. Dysregulation of these interactions can lead to disruption of retinal architecture and impairment of vision.

Abstract

In the embryonic nervous system, developing axons can be guided to their targets by diffusible factors secreted by their intermediate and final cellular targets. To date only one family of chemoattractants for developing axons has been identified. Grafting and ablation experiments in fish, amphibians, and birds have suggested that spinal motor axons are guided to their targets in the limb in part by a succession of chemoattractants made by the sclerotome and by the limb mesenchyme, two intermediate targets that these axons encounter en route to their target muscles. Here we identify the limb mesenchyme-derived chemoattractant as hepatocyte growth factor/scatter factor (HGF/SF), a diffusible ligand for the c-Met receptor tyrosine kinase, and we also implicate HGF/SF at later stages as a muscle-derived survival factor for motoneurons. These results indicate that, in addition to functioning as a mitogen, a motogen, and a morphogen in nonneural systems, HGF/SF can function as a guidance and survival factor in the developing nervous system.

Abstract

Although glial cells ensheath synapses throughout the nervous system, the functional consequences of this relationship are uncertain. Recent studies suggest that glial cells may promote the formation of synapses and help to maintain their function by providing nerve terminals with energy substrates and glutamate precursors.

Abstract

Although ciliary neurotrophic factor (CNTF) is a potent survival factor for many types of neurons and glial cells in vitro, there is currently no evidence that it participates in normal development. Here we show that CNTF greatly enhances the rate of oligodendrocyte generation. Proliferation of oligodendrocyte precursor cells purified from rodent optic nerves and cultured in platelet-derived growth factor-containing medium is significantly increased by CNTF. Similarly, the number of proliferating oligodendrocyte precursor cells in developing optic nerves of transgenic mice lacking CNTF is decreased by up to threefold and the number of oligodendrocytes is transiently decreased; proliferation is restored to normal by the delivery of exogenous CNTF into the developing optic nerve. Both oligodendrocyte number and myelination ultimately attain wild-type values in CNTF-deficient adult mice, indicating that CNTF is not necessary for either oligodendrocyte differentiation or myelination, although it normally accelerates oligodendrocyte development by enhancing the proliferation of oligodendrocyte precursor cells.

Abstract

Although ciliary neurotrophic factor (CNTF) is a potent survival factor for many types of neurons and glial cells in vitro, there is currently no evidence that it participates in normal development. Here we show that CNTF greatly enhances the rate of oligodendrocyte generation. Proliferation of oligodendrocyte precursor cells purified from rodent optic nerves and cultured in platelet-derived growth factor-containing medium is significantly increased by CNTF. Similarly, the number of proliferating oligodendrocyte precursor cells in developing optic nerves of transgenic mice lacking CNTF is decreased by up to threefold and the number of oligodendrocytes is transiently decreased; proliferation is restored to normal by the delivery of exogenous CNTF into the developing optic nerve. Both oligodendrocyte number and myelination ultimately attain wild-type values in CNTF-deficient adult mice, indicating that CNTF is not necessary for either oligodendrocyte differentiation or myelination, although it normally accelerates oligodendrocyte development by enhancing the proliferation of oligodendrocyte precursor cells.

Abstract

The signaling mechanisms that control the survival of CNS neurons are poorly understood. Here we show that, in contrast to PNS neurons, the survival of purified postnatal rat retinal ganglion cells (RGCs) in vitro is not promoted by peptide trophic factors unless their intracellular cAMP is increased pharmacologically or they are depolarized by K+ or glutamate agonists. Long-term survival of most RGCs in culture can be promoted by a combination of trophic factors normally produced along the visual pathway, including BDNF, CNTF, IGF1, an oligodendrocyte-derived protein, and forskolin. These results suggest that neurotransmitter stimulation and electrical activity enhance the survival of developing RGCs and raise the question of whether the survival control mechanisms of PNS and CNS neurons are different.

Abstract

We draw the following tentative conclusions from our studies on programmed cell death (PCD): (i) the amount of normal cell death in mammalian development is still underestimated; (ii) most mammalian cells constitutively express the proteins required to undergo PCD; (iii) the death programme operates by default when a mammalian cell is deprived of signals from other cells; (iv) many normal cell deaths may occur because cells fail to obtain the extracellular signals they need to suppress the death programme; and (v) neither the nucleus nor mitochondrial respiration is required for PCD (or Bcl-2 protection from PCD), raising the possibility that the death programme, like mitosis, is orchestrated by a cytosolic regulator that acts on multiple organelles in parallel.

Abstract

The timing of oligodendrocyte differentiation is thought to depend on an intrinsic clock in oligodendrocyte precursor cells that counts time or cell divisions and limits precursor cell proliferation. We show here that this clock mechanism can be separated into a counting component and an effector component that stops cell proliferation: whereas the counting mechanism is driven by mitogens that activate cell-surface receptors, the effector mechanism depends on hydrophobic signals that activate intracellular receptors, such as thyroid hormones, glucocorticoids and retinoic acid. When purified oligodendrocyte precursor cells are cultured at clonal density in serum-free medium in the presence of mitogens but in the absence of these hydrophobic signals, the cells divide indefinitely and do not differentiate into postmitotic oligodendrocytes. In the absence of mitogens, the precursor cells stop dividing and differentiate prematurely into oligodendrocytes even in the absence of these hydrophobic signals, indicating that these signals are not required for differentiation. The levels of these signals in vivo may normally regulate the timing of oligodendrocyte differentiation, as the maximum number of precursor cell divisions in culture depends on the concentration of such signals and injections of thyroid hormone into newborn rats accelerates oligodendrocyte development. As thyroid hormone, glucocorticoids and retinoic acid have been shown to promote the differentiation of many types of vertebrate cells, it is possible that they help coordinate the timing of differentiation by signalling clocks in precursor cells throughout a developing animal.

Abstract

During the development of the vertebrate nervous system, up to 50 percent or more of many types of neurons normally die soon after they form synaptic connections with their target cells. This massive cell death is thought to reflect the failure of these neurons to obtain adequate amounts of specific neurotrophic factors that are produced by the target cells and that are required for the neurons to survive. This neurotrophic strategy for the regulation of neuronal numbers may be only one example of a general mechanism that helps to regulate the numbers of many other vertebrate cell types, which also require signals from other cells to survive. These survival signals seem to act by suppressing an intrinsic cell suicide program, the protein components of which are apparently expressed constitutively in most cell types.

Abstract

We have shown previously that oligodendrocytes and their precursors require signals from other cells in order to survive in culture. In addition, we have shown that about 50% of the oligodendrocytes produced in the developing rat optic nerve normally die, apparently in a competition for the limiting amounts of survival factors. We have hypothesized that axons may control the levels of such oligodendrocyte survival factors and that the competition-dependent death of oligodendrocytes serves to match their numbers to the number of axons that they myelinate. Here we test one prediction of this hypothesis - that the survival of developing oligodendrocytes depends on axons.We show that oligodendrocyte death occurs selectively in transected nerves in which the axons degenerate. This cell death is prevented by the delivery of exogenous ciliary neurotrophic factor (CNTF) or insulin-like growth factor I (IGF-1), both of which have been shown to promote oligodendrocyte survival in vitro. We also show that purified neurons promote the survival of purified oligodendrocytes in vitro.These results strongly suggest that oligodendrocyte survival depends upon the presence of axons; they also support the hypothesis that a competition for axon-dependent survival signals normally helps adjust the number of oligodendrocytes to the number of axons that require myelination. The identities of these signals remain to be determined.

Abstract

Oligodendrocytes myelinate axons in the vertebrate central nervous system. It would, therefore, make sense if axons played a part in controlling the number of oligodendrocytes that develop in a myelinated tract. Although oligodendrocytes themselves normally do not divide, the precursor cells that give rise to them do. Here we show that the proliferation of oligodendrocyte precursor cells in the developing rat optic nerve depends on electrical activity in neighbouring axons, and that this activity-dependence can be circumvented by experimentally increasing the concentration of platelet-derived growth factor, which is present in the optic nerve and stimulates these cells to proliferate in culture. These findings suggest that axonal electrical activity normally controls the production and/or release of the growth factors that are responsible for proliferation of oligodendrocyte precursor cells and thereby helps to control the number of oligodendrocytes that develop in the region.

Abstract

We have recently found that about 50% of newly formed oligodendrocytes normally die in the developing rat optic nerve. When purified oligodendrocytes or their precursors are cultured in the absence of serum or added signalling molecules, they die rapidly with the characteristics of programmed cell death. This death is prevented either by the addition of medium conditioned by cultures of their normal neighboring cells in the developing optic nerve, or by the addition of platelet-derived growth factor (PDGF) or insulin-like growth factors (IGFs). Increasing PDGF in the developing optic nerve decreases normal oligodendrocyte death by up to 90% and doubles the number of oligodendrocytes, suggesting that this normally occurring glial cell death might result from a competition for limiting amounts of survival signals. These results suggest that competition for limiting amounts of survival factors is not confined to developing neurons, and raise the possibility that a similar mechanism may be responsible for some naturally occurring cell deaths in nonneural tissues.

Abstract

Dead cells are observed in many developing animal tissues, but the causes of these normal cell deaths are mostly unknown. We show that about 50% of oligodendrocytes normally die in the developing rat optic nerve, apparently as a result of a competition for limiting amounts of survival signals. Both platelet-derived growth factor and insulin-like growth factors are survival factors for newly formed oligodendrocytes and their precursors in culture. Increasing platelet-derived growth factor in the developing optic nerve decreases normal oligodendrocyte death by up to 90% and doubles the number of oligodendrocytes in 4 days. These results suggest that a requirement for survival signals is more general than previously thought and that some normal cell deaths in nonneural tissues may also reflect competition for survival factors.